Indian Journal of Experimental Biology Vol. 43, November 200S, pp. 1016-1022

Review Article

Does human sperm nuclear DNA integrity affect embryo quality?

Silpi Acharyya l, Sagarika Kanjilaf & Asok K Bhattacharyya l *

'Department of Biochemistry, University of Calcutta, 3S, Ballygunge Circular Road, Kolkata 700 019, India.

2Department of Dermatology & Veterinary Pathobiology, University of Minnesota, St. Paul, MNSS108, USA.

Abnormalities in the male genome are a clear potential reason for post fertilization failure. Male infertility may arise due to high levels of loosely packaged chromatin and damaged DNA. The achievement of a correct chromatin packaging level is essential for successful fertilization. The chromatin contained in the nuclei of mammalian spermatozoa is an extremely compact and stable structure. The reports on mammalian spermatozoa indicate that available volume is insufficient to contain sperm chromatin packed in nucleosoll1e like structure and thus is organized in a special way . Different unique properties of sperm DNA like high degree of inertness and stability, absence of transcription, replacement of somatic histone by protamine etc have made the study of sperm chromatin more interesting. Increased levels of sperm nuclear DNA damage exist in infertile men with abnormal sperm parameters (i .e. concentration, motility and morphology), and various assay techniques have been developed to evaluate sperm chromatin maturitylDNA integrity. These assays are based on the facts that defects in chromatin structure have been shown to lead to increased DNA instability and sensitivity to denaturing stress. DNA integrity in the sperm is essential for the accurate and successful transmission of genetic information. Importance of sperm DNA has also become more obvious in the context of assisted reproductive techniques. While recent advances in assisted reproductive technologies have made possible and practical for many infertile men to become father, the risk of transmission of genetic mutation to the offspring, however, still remains. Further research is necessary to devise techniques for identification and selection of sperm with undamaged DNA for ICSI or to remove sperm with damaged DNA from the semen sample to improve the pregnancy outcome in ICSI.

Keywords: DNA, Embryo, Human sperm, Nuclear DNA, Sperm

VariOllS biochemical and physicochemical charac­teristic features of chromatin from a large number of sources are reported in the literature, but the information on the properties of chromatin from mammalian spermatozoa is limited. Sperm chromatin is a highly organized compact structure consisting of DNA and heterogeneous nucleoproteins. Sperm DNA is known to contribute one half of the genomic material to the offspring and thus normal sperm genetic material is required for fertilization, embryo development. and for postnatal child well being. The quality of sperm DNA is very important in main­taining the reproductive potential of men. It is important to understand that infertile men who have genetic problems can pass this to their offspring and may give rise to birth defects. Male infertile patients can have anomalies in their sperm nuclei displaying high levels of loosely packaged chromatin and damaged DNA. Packaging of the sperm chromatin may serve to reprogram me the paternal genome and

*Correspondent author Phone: +91 -33-2466 1264, Fax: +91-33-24764419 Email: ashok.bhattacharyya@rediffmail.com

set the appropriate genes to be expressed in the early stages of embryo development. Thus, the achievement of a correct chromatin packaging level seems essential to express fully the fertilizing capability of sperm 1.

Stable DNA that is capable of decondensation during the appropriate time in the fertilization process is also one of the essential criteria for considering a sper­matozoon to possess fertilizing abilitl. Abnormalities in the male genome however are a clear potential reason for post fertilization failure3

.s.

The importance of sperm DNA has become more obvious in the context of assisted reproductive techniques (ART), which has gained tremendous importance in the treatment of infertile couples6

. It is documented that a significant proportion of infertile men with azoospermia and severe oligozoospermia has a genetic aetiology for reproductive failure. While recent advances in assisted reproductive technologies have made possible and practical for many infertile men to become father, the risk of transmission of genetic mutation to the offspring still remains. The main disadvantage of ART is that these techniques bypass the natural selection barrier that is present

ACHARYY A et al.: HUMAN SPERM NUCLEAR DNA INTEGRITY 1017

throughout the male and female reproductive tract until the sperm enters the oocyte7

. In the contem­porary practice of infertility management, intrauterine insemination (lUI), intra-cytoplasmic sperm injection (ICSI) and in-vitro fertilization (IVF) are the commonly used assisted reproductive technologies. With ART, sperm with abnormal genomic material can reach the oocyte with minimal effort in lUI and IVF and with almost no effort in ICSI. ICSI introduces the male genome directly into the oocyte and since spermatozoon is selected for this purpose based only on motility and gross morphology, concern has been expressed that such method could introduce a defective paternal genome8

-11 . Thus,

sperm DNA became the study of extensive research -during the last two decades.

Sperm chromatin structure The chromatin contained m the nuclei of

mammalian spermatozoa is an extremely compact and stable structure. Tight packaging of the chromatin minimizes volume of the sperm nuclei for transport through the female system and for protection of genetic material from any kind of damage. To achieve this high degree of packaging, sperm DNA is supposed to interact with the protein part in a very special way, which differs markedly from that of somatic cell nuclei where DNA is coiled around histone octamers to form nUcleosome12.

There is significant evidence suggesting that sperm nuclei are essentially crystalline aggregates of nucleoprotein complexes 13. Freeze fracture studies of spermatozoa in bulls 14, rabbits l5 and rat16 indicate that mammalian sperm nuclei seem to be composed of parallel stacks of lamellar sheets oriented to the sperm's long axis . Fluorescence in situ hybridization (FISH) suggests that a discrete nodular structure of - 300 nm diameter may represent the basic packaging unit of mammalian sperm chromatin 17. The bull, mouse and rat sperm chromatin may have a nodular organization, as shown by Atomic Force Microscopy (AFM)18.

Organization of chromatin for packaging in human spermatozoon takes place at four different levels: chromosomal anchoring, which refers to the attachment of DNA to nuclear annulus; formation of the DNA loop domains as the DNA attaches to the newly added nuclear matrix; replacement of histones by protamines, which condense the DNA into compact doughnuts; and chromosomal positioning12.

The fundamental packaging unit of sperm chromatin is a toroid with approximately 900A outer diameter, 200A thickness and 150A diameter hole. Each toroid contains 60 kb of DNA and is linked to other toroids by uncoiled DNA stretches 17. Chromatin organization in sperm nuclei is maintained during sperm condensation by tight interactions with the nuclear matrix at fixed sites including formation of individual toroid shaped DNA loop structures.

Protamines in the sperm chromatin are highly basic proteins about half the size of a typical histone. About 55-79% of its amino acid residues are arginine (permitting strong DNA binding) <,.ad a significant number of cysteine residues (participate in chromatin compaction by formation of intra and inter disulphide protamine linkage) are important in final stages of sperm nuclear maturation 19. While the histone-DNA structure is highly conserved in most eukaryotic organism, protamines show a high degree of specificity2o. Not all species replace histones in the sperm nucleus during spermiogenesis, some species like Carp21 retain histones as typical proteins in the sperm nuclei. Other species like human retain up to 15% of their original histone content22. In some mammalian species like rat, mouse and sheep, histones are first replaced by a "transition protein" and finally by one or more species of protamines23. The extent of replacement of histone by protamine in different animal species is summarized in Table 1.

Sperm chromatin vs somatic cell chromatin­Sperm chromatin is packed into a tight, almost crystalline status that is at least 6 times more condensed than mitotic chromosomes. It occupies almost the entire nuclear volume whereas somatic cell DNA fills the nucleus l7 only partly. Sperm nuclei do not have the volume required for the type of packaging present in somatic cells, because packaging the DNA even in a single closely packed nucleosome would require 9.9jlm3

, which is more than twice the volume of an average sperm nucleus 12.

Table 1--N ature of protein in sperm chromatin of different mammals

Different animals species having sperm chromatin with Histone Protamine Transition protein

Mice27, Boar24, Rabbit15, BuU25, Ram25, Rat16, Human*22 Ram25, Stallion26, Mice27

, Human#22 Mice27

, Rat16, Boar24, Human27

* Only 15% histone exists in human sperm chromatin # Presence of transition protein is doubtful

1018 INDIAN J EXP BIOL, NOVEMBER 2005

Sperm DNA characteristics The DNA of spermatozoa is so tightly complexed

to the nuclear protein that transcription is almost impossible28. Different unique properties of sperm DNA like high degree of inertness and stability, absence of transcription, replacement of somatic histone by protamine etc have made the study of sperm chromatin more interesting.

The DNA content is approximately 1.36xlO-9 mg in a single nucleus of human spermatozoa. In other mammals like bull, rabbit and ram, the DNA measures 3.22xlO-9 mg, 3.2xlO-9 mg and 2.93xlO-9

mg per sperm nucleus, respectively28. Some properties of DNA from mammalian sperm nuclei are summarized in Table 2.

Sperm nuclear DNA damage Several studies have shown increased levels of

sperm nuclear DNA damage in infertile men with abnormal sperm parameters (i .e. concentration, motility and morphology) 32. Although the extent of DNA damage is closely related to sperm function and male infertiliti3, the origin of such damage is still largely controversial. Several factors that may give rise to DNA damage are as follows:

Oxidative stress-This is caused by an imbalance between production of reactive oxygen species (ROS) and antioxidant capacity34. ROS may lead to chromatin cross-linking35, DNA strand breaks36

, DNA base oxidation37, chromosome deletions, dicentrics, and sister chromatid exchanges5. Oxidative stress has also been correlated with apoptosis38 and high frequencies of single and double DNA strand breaks5.

Defects in chromatin packaging-Immature spermatozoa have high levels of DNA damage and ROS production, and are likely to have alterations in protamination and chromatin packaging39.

Table 2--Characteristics of sperm DNA in some mammals

Example SEerm DNA Ero~rt:z: Content'9.3U Melting Sedimen- Hyper- Bouyant

(x 10-9 tempe- tation chromic densit/ I

mg/nucleus) rature31 velocity31 effece l (mice) Tm (0C) (s) (~260nm;

in %)

Man 1.35-1.37 89.0 16.5 30 1.701 2.45-2.86#

Bull 3.22 87-90 18.1-23.4 33-37 1.701 Rabbit 3.1 88 12.8 33

# S~rm DNA content in oligozoosEermie semen samEle

Apoptosis-Apoptosis controls the overproduction of male gametes and restricts normal proliferation levels so that they do not surpass the supportive capacity of sertoli cells 40. Incomplete endogenous nuclease activity creates and ligates nicks during spermiogenesis41 .

Other causes-Increased DNA damage in sperm from leukocytospermic semen sample has also been

d42 E . . I reporte . xposure to tOXIC agents, environmenta or occupational hazards can lead to abnormal reproduc­tive outcomes by altering the integrity of genetic material at chromosome or DNA level, in male germ cells43. Even though a large amount of data is not available, smoking, alcohol, caffeine consumption, and anticancer drugs have been reported to cause aneuploidy in human sperm44. Sperm preparation techniques involving centrifugal pelleting have also been considered to result in sperm DNA damage and subsequent failure in pregnancy in some cases 10.

Evaluation of sperm DNA damage Several assays have been developed to evaluate

sperm chromatin maturitylDNA integrity. These assay methods were based on the facts that defects in the chromatin structure have been shown to lead to increased DNA instability and senSItIVity to denaturing stress20. In addition, spermatozoa from infertile men have a higher frequency of chromosomal abnormalities45 , poor DNA packaging quality46, increased DNA strand breaks47, and susceptibility to acid-induced DNA denaturation in situ in comparison to spermatozoa from fertile men48. Table 3 sum­marizes various techniques employed in identifying DNA damage in spermatozoa.

Effect of sperm DNA damage on male infertility DNA integrity in the sperm is essential for the

accurate and successful transmission of genetic information and for the maintenance of good health in future generation. The function as well as replication of every cell are dependent on its DNA. In sperm cells, DNA damage may carry mutations into the next generation or result in male infertility57. The increased sensitivity to sperm DNA damage is probably due to failed chromatin condensation, which makes the DNA more accessible to damage58-60

. Presence of apoptotic DNA fragmentation in ejaculated spermatozoa may also be due to defects in spermatogenesis61 . The increased presence of these anomalies in males with abnormal sperm parameters put the population of patients being treated by ART, in particular ICSI at

ACHARYYA et at. : HUMAN SPERM NUCLEAR DNA INTEGRITY 1019

Table 3---Various DNA damage detection techniques

Instrument involved Assay method

Fluorescence microscope In situ nick translation49

Acridine orange staining50

Detection principle

Single-strand DNA breaks

Differentiation between single and double stranded DNA

Alkaline single-cell gel electrophoresis (Comet assay) 51

Evaluation of DNA integrity, single and double-strand DNA breaks

Sperm chromatin decondensation52

Chromamycin A353

Flow cytometer TUNEL assay54

Production of characteristic DNA decondensation halo with non­fragmented DNA from intact spermatozoa

Indirect visualization of nicked, denatured DNA

DNA fragmentation, single- and double­strand DNA breaks

Optical microscope

Sperm chromatin structure assay (SCSA) 55

Toluidene blue stain56

Acid DNA denaturation

Incorporation of stain in the damaged dense chromatin

HPLC 8-oxo-7,8 dihydro-2 deoxyguanosine (8-0H-dG) 38

HPLC with electrochemical detection

greater risk. Whether DNA damaged sperm can impair the process of fertilization or embryo development is not clear.

There may be some controversies about the impact of high sperm DNA fragmentation on the fertilization rate, but there is a wider agreement concerning their negative effects on embryo development and preg­nancy rate62

•63

. However, Ahmadi & Ng64 have reported that under normal circumstances, low levels of damage can be repaired by the oocyte, but DNA damage that is beyond repair will result in apoptosis and fragmentation of the early embryo or morbidity in later life65

• In case of genetic damage as judged by Comet assay, a few embryos may reach the blastocyst stage, but natural selection ensures that most of them will abort before growing to term66

. Tomsu et aI.66

have also attempted to establish a correlation between sperm DNA integrity and cleavage rates and embryo qUality.

Correlation between sperm DNA damage and Assisted Reproductive Techniques (ART)

In the era of ART, the integrity of sperm nuclear DNA is of paramount concern for the successful transmission of a competent paternal genome to the oocyte. In the contemporary practice of infertility management, lUI often precedes the costly and rigorous options of ARTs, such as IVF and/or ICSI. Duran et a1. 67 have reported that sperm DNA quality

could predict the lUI outcome and their observation state that pregnancy is impossible when DNA fragmentation is more than 12%. Earlier Larson et al. 52 have shown that in case of ICSI, DNA fragmentation in more than 27% sperm cannot give rise to pregnancy. Similar observation was made by Benchaib et aI.68 who have shown that fertilization is impossible in case of DNA damage of more than 20%. On the contrary, Tomlinson et al. 69 state that there is no correlation of sperm DNA damage with fertilization rate in patients undergoing IVF procedure. In contrast to this observation, Host et al. 70

in their study have found that sperm DNA damage is associated with semen parameters, fertilization rate, and IVF outcome, but no correlation has been found between the nuclear DNA fragmentation rate and the fertilization rate in ICSI patients. Other reports indicate that in ART procedure, sperm DNA fragmen­tation is negatively correlated with fertilization success7

!, embryo cleavage rate63• 65 and with embryo

development72. The sperm head and tail DNA parameters are also known to be useful in predicting fertilized embryo qUality66.

It remains, however, unclear whether ART can compensate for poor chromatin packaging and/or DNA damage or that sub-optimal chromatin integrity may contribute to the poor «20%) implantation rate seen in most ART patients68

• The fact that the fertilization and pregnancy rates were surprisingly

1020 INDIAN J EXP BIOL, NOVEMBER 2005

high after ICSI regardless of the severity of the sperm defect73 has subsequently raised debate on the possibility of using ICSI to force fertilization by abnormal spermatozoa. Reports indicate that the levels of embryo loss are similar following IVF and ICSI67

• However, Sanchez et al.74 have found that the miscarriage rate following ICSI is greater than that expected with conventional IVF, possibly reflecting the use of genomically compromised spermatozoa that confer irrepairable DNA damage to the embryo and caused its subsequent abortion. A significantly high percentage of DNA strand break is also seen in surgically extracted spermatozoa from the epididymis and testicular tissue during ICSI trials for men with obstructive azoospermia75

•

Role of standard semen parameters in the evaluation of sperm quality has become debatable with the advent of ART procedures. These procedures bypass the natural selection mechanisms, and increase the chance, that abnormal genomic material will fertilize an oocyte. Extensive data on the relationship between sperm DNA damage and post ART outcome have shown correlation of sperm DNA fragmentation with post fertilization failure. Some studies have found no association between the levels of DNA damage and fertilization rate as well as post fertilization embryo development, but most of the studies have shown a negative trend with fertility outcome parameters after ART. Many ICSI studies have found a lack of correlation between fertilization rates and DNA damage suggesting that ICSI allows sperm with damaged DNA to fertilize oocyte. There are controversies regarding the rate of embryo loss in patients undergoing ART for damage in DNA of their sperm, but it can be said that there is chance of introduction of a defective paternal genome in ICSI. Thus, further research is necessary to devise techniques for ~dentification of sperm with undamaged DNA or to remove sperm with damaged DNA from the semen sample to improve the pregnancy outcome in ICSI. It is also important to standardize the protocols used to detect chromosomal abnormalities and DNA damage so that they can be routinely used in the laboratories prior to ART. Detection of sperm nuclear DNA integrity is, thus, a potential tool for evaluation of semen samples prior to their use in ART.

Acknowledgement The authors · gratefully acknowledge Council of

Scientific & Industrial Research (CSIR), New Delhi,

India [Project No. F.No.9/28(550)/2001-EMR-I] for providing financial support.

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