Given the cost of this form of treatment, and the fact that children born as a consequence of ART stand a 30–40% increased risk of birth defects [3],

the current widespread use of assisted conception may constitute the beginnings of a serious public health problem.

Full Text
Expert Review of Obstetrics & Gynecology
May 2008, Vol. 3, No. 3, Pages 267-271
(doi:10.1586/17474108.3.3.267)



Just how safe is assisted reproductive technology for treating male factor infertility?
R John Aitken
Sections: ChooseINTRODUCTIONThe male factorParental ageEnvironmental pollutionErrors of endogenous meta...Electromagnetic radiation...Epigenetic damageIs ART a dangerous form o...References





INTRODUCTION ChooseTop of pageINTRODUCTION The male factorParental ageEnvironmental pollutionErrors of endogenous meta...Electromagnetic radiation...Epigenetic damageIs ART a dangerous form o...References

Assisted reproductive technology (ART) has been responsible for the birth of over 3 million babies since the delivery of Louise Brown in the UK 28 years ago. Currently, one in 80–100 children born in the USA, one in 50 born in Sweden, one in 40 born in Australia and one in 24 born in Denmark are the product of this form of treatment. In 2003, more than 100,000 in vitro fertilization (IVF) cycles were reported from 399 clinics in the USA, resulting in the birth of more than 48,000 babies [1,101]. Worldwide, this figure has now exceeded 200,000 births per annum [2] and is continuing to rise. Indeed, it is a biological certainty that the more ART is used in one generation, the more it will be needed in the next. Given the cost of this form of treatment, and the fact that children born as a consequence of ART stand a 30–40% increased risk of birth defects [3], the current widespread use of assisted conception may constitute the beginnings of a serious public health problem.


The male factor ChooseTop of pageINTRODUCTIONThe male factor Parental ageEnvironmental pollutionErrors of endogenous meta...Electromagnetic radiation...Epigenetic damageIs ART a dangerous form o...References

There is general agreement that the two major reasons for patient referral to assisted conception programs are increased maternal age and male subfertility. The former can be easily reversed by public awareness and a change in social attitudes to family planning. However, the latter is a more intractable problem, as we have little or no understanding about the origins of this pathology. As a result, rational treatment or prevention of male infertility is all but impossible.

The importance of the male factor in human infertility has been highlighted by recent analyses of population trends in Denmark. This population has witnessed a steady decline in fertility rates in recent years, which is being addressed by increasing reliance on ART [4]. At the present time, 21% of young Danish men exhibit semen quality (in terms of sperm count and morphology) that falls below the internationally accepted thresholds of normality set by the WHO [5]. Moreover, it has been suggested that this situation is getting worse with the passage of time and, according to a recent publication [6]:

‘we may now have reached a level where semen quality of a significant segment of men in the population is so poor that it may contribute to the current widespread use of assisted reproduction’.

Although Denmark affords a particularly striking example of secular trends in male reproduction, semen quality in human males is notoriously poor. Indeed, it is a feature of the human condition, with at least one in 20 men in developed countries suffering from some level of infertility [7]. Most men produce sufficient numbers of spermatozoa to fertilize an egg in vivo; however, the gametes they generate have lost their biological potential for fertilization and the support of normal embryonic development. An important characteristic of these defective spermatozoa is a high level of DNA damage, which is, in turn, correlated with poor fertility, high rates of miscarriage and an increased incidence of disease in the offspring, including childhood cancer [8].

The use of such DNA-damaged spermatozoa in ART is thought to be a major contributor to the increased incidence of birth defects and other diseases seen in children conceived in vitro. Specifically, it has been proposed that the DNA damage brought into the fertilized egg by the spermatozoon may increase the mutational load carried by the embryo as a consequence of the aberrant or incomplete repair of this damage in the interval between fertilization and initiation of the first cleavage division [9,10]. Experimental verification of this relationship between DNA damage in the fertilizing sperm and embryo development has recently been secured in an animal model [11]. In these studies, intracytoplasmic sperm injection (ICSI) was performed in mice with fresh or DNA-damaged spermatozoa. Use of the latter was associated with poor preimplantation development and a reduction in the number of live births. Postnatal examination of the progeny revealed that the use of DNA-damaged spermatozoa in ICSI was associated with behavioral defects (increased anxiety, lack of habituation pattern, deficit in short-term spatial memory and age-dependent hypolocomotion in an open field test), the appearance of mesenchyme tumors, premature aging and a shortened lifespan. These results are supported by clinical data [8] and have profound implications for the safety of ICSI, which must frequently involve the use of DNA-damaged spermatozoa [12]. Currently, both the nature of this genetic damage and its origins are a matter of intense investigation. In terms of etiology, the ensuing paragraphs summarize data suggesting that paternal age, environmental toxicants, errors of endogenous metabolism and exposure to electromagnetic radiation are all potential contributors to DNA damage in the male germ line.



Parental age ChooseTop of pageINTRODUCTIONThe male factorParental age Environmental pollutionErrors of endogenous meta...Electromagnetic radiation...Epigenetic damageIs ART a dangerous form o...References

As men age they do not lose their capacity to generate spermatozoa; however, the quality of these gametes deteriorates. This change can be visualized as an age-dependent increase in DNA fragmentation in spermatozoa [13,14]. Paternal age is also widely recognized as a key factor in the etiology of dominant genetic diseases, such as Apert syndrome or achondroplasia [15]. Furthermore, genetic damage to the spermatozoa of aging males is thought to contribute to the etiology of more complex polygenic conditions such as autism, spontaneous schizophrenia and epilepsy [8]. Since older men tend to be married to older women it is significant that as oocytes age in the ovary, they suffer the depletion of several key genes involved in protection against oxidative stress and the maintenance of DNA integrity, including genes with a probable role in DNA repair [16]. Thus, age-related changes to the integrity of DNA in the spermatozoa are compounded by age-related declines in the oocytes’ capacity for DNA stabilization and repair. In combination, these factors could well make a significant contribution to the elevated incidence of birth defects associated with assisted conception therapy. Whichever way you look at it, aging and reproduction are incompatible bedfellows.



Environmental pollution ChooseTop of pageINTRODUCTIONThe male factorParental ageEnvironmental pollution Errors of endogenous meta...Electromagnetic radiation...Epigenetic damageIs ART a dangerous form o...References

An impact of environmental pollutants on DNA integrity in spermatozoa has been known for some time. For example, men who smoke heavily produce spermatozoa suffering from high levels of oxidative DNA damage. This does not necessarily impair the capacity of these cells for fertilization, however, it does impact upon the subsequent ability of the fertilized egg to develop normally. As a result, the children of heavy smokers stand a four- to fivefold increased chance of developing childhood cancer: a fact that is not often appreciated in the antismoking debate [9].

Recently, exposure of mice to particulate air pollution in an urban/industrial location has also been shown to induce high levels of DNA damage in spermatozoa [17]. Analyses of young men exposed to high levels of air pollution as a result of excessive coal combustion during Eastern European winters have substantiated these results in a clinical context [18]. Similarly, toxicological studies have demonstrated elevated levels of DNA damage in human spermatozoa, which are linked to the presence of metabolites of insecticides or persistent organochlorine pollutants in blood or urine [19,20]. Exposure to environmental endocrine disruptors, such as nonylphenol [21], as well as heavy metals [22], have also been demonstrated to induce oxidative DNA damage in human spermatozoa. Further resolution of the kinds of environmental pollutant that might be damaging to human spermatozoa is clearly needed. Elucidation of the significance of enzyme polymorphisms in defining an individual’s susceptibility to toxicant exposure is also required, as exemplified by a recent study demonstrating that men who are homozygous null for glutathione-S-transferase M1 are more likely to respond to air pollution with high levels of DNA damage in their spermatozoa than men possessing this isoform [23].



Errors of endogenous metabolism ChooseTop of pageINTRODUCTIONThe male factorParental ageEnvironmental pollutionErrors of endogenous meta... Electromagnetic radiation...Epigenetic damageIs ART a dangerous form o...References

Induction of DNA fragmentation in human spermatozoa is not solely due to exposure to environmental toxins, it can also result from errors of endogenous metabolism. An extremely important observation in this context is a recent preliminary report indicating that young male patients suffering from diabetes mellitus exhibit high levels of DNA damage in their spermatozoa [24]. These results have been confirmed in animal studies demonstrating that the experimental induction of diabetes in male mice is associated with oxidative stress and a postmeiotic genotoxic effect reflected in high rates of embryonic resorption in mated females [25]. Our laboratory has also demonstrated that endogenously generated estrogens, particularly catechol estrogens, can have a profound effect on DNA integrity in human spermatozoa, as a consequence of their inherent redox cycling activity [26]. Such studies reinforce the generally held view that most endogenously generated DNA damage in human spermatozoa is a consequence of oxidative stress [8,28]. If this is the case, then any ion (lead or cadmium), organic compound (phthalate ester), enzyme (NADPH oxidase), organelle (mitochondria) or cell (neutrophil), capable of generating reactive oxygen species in the vicinity of human spermatozoa is potentially capable of contributing to DNA damage in the male germ line [8–10,22,28]. In addition to oxidative damage, it is possible that in some patient’s sperm DNA is cleaved by the sequential action of topoisomerase IIB and an uncharacterized nuclease in a process analogous to apoptosis in somatic cells [29]. The relative significance of nuclease- and free radical-mediated mechanisms in the cleavage of sperm DNA, is a key issue that awaits resolution.



Electromagnetic radiation ChooseTop of pageINTRODUCTIONThe male factorParental ageEnvironmental pollutionErrors of endogenous meta...Electromagnetic radiation... Epigenetic damageIs ART a dangerous form o...References

Various forms of electromagnetic radiation are also known to have a detrimental effect on DNA integrity in the male germ line. A classic example is heat. The scrotum is designed to maintain the testes and epididymis slightly below core body temperature. It has been known for some time that elevated testicular temperature impairs spematogenesis. However, recent data have also indicated the ability of mild scrotal heat stress (42°C for 30 min) to induce DNA damage in epididymal mouse spermatozoa [30]. Radiofrequency electromagnetic radiation has also been demonstrated to induce DNA damage in epididymal sperm in animal models [31] and there are some reports of mobile phone exposure having a detrimental effect on semen quality in men [32]. Thus, any practice that elevates testicular temperature, such as wearing clothes or a sedentary occupation, or exacerbated exposure to other forms of electromagnetic radiation, such as excessive mobile phone use, are possible contributors to DNA damage in human spermatozoa.



Epigenetic damage ChooseTop of pageINTRODUCTIONThe male factorParental ageEnvironmental pollutionErrors of endogenous meta...Electromagnetic radiation...Epigenetic damage Is ART a dangerous form o...References

In some couples, the damage brought into the oocyte by the fertilizing spermatozoon may be epigenetic rather than genetic. These epigenetic factors are reviewed in an article in this edition of Expert Review of Obstetrics and Gynecology [31] and include: a functional centrosome to regulate cell division in the embryo; an appropriate pattern of chromatin remodeling; an appropriate population of mRNA and miRNA species that are carried into the zygote by the fertilizing spermatozoon and may play a role in the regulation of early embryonic development; and an appropriate pattern of DNA methylation. There are several recent papers indicating that the DNA methylation profile is dramatically altered in the spermatozoa of infertile men and we already know that the incidence of imprinting defects is elevated in children born as a result of ART [31,32]. The importance of epigenetic defects in the male germ line has recently been highlighted by analyses of vinclozolin, a fungicide used in the wine-making industry [33]. Transient embryonic exposure to vinclozolin in utero resulted in the birth of male offspring exhibiting a spermatogenenic defect. This defect was epigenetic in origin and was vertically transmitted through at least four generations.



Is ART a dangerous form of therapy? ChooseTop of pageINTRODUCTIONThe male factorParental ageEnvironmental pollutionErrors of endogenous meta...Electromagnetic radiation...Epigenetic damageIs ART a dangerous form o... References

Given the evidence that both IVF and ICSI are associated with a significant increase in birth defects, should ART be regarded as safe? On one hand, there is no denying that ART, and particularly ICSI, is an effective form of treatment for infertility. After 12 months approximately 90% of couples submitting to this form of therapy walk away with a baby. Furthermore, even though the risk of birth defects is significantly elevated following ART, the incidence is still relatively rare and should decrease as the field moves towards single-embryo transfers, thereby eliminating complications created by multiple births. Moreover, several clinical groups have trumpeted their ability to successfully perform ART in couples where the male partner’s spermatozoa exhibit high levels of DNA damage, without any obvious consequences as far as the health and wellbeing of the offspring are concerned [34]. These and other data tell us that even if DNA-damaged spermatozoa are used for assisted human conception, the risk of generating a visible phenotypic change in the offspring is extremely low.

However, we should also recognize that the absence of a pathological phenotype in the vast majority of children born as a result of ART, does not mean that the genome has not been damaged, or that the damage will not emerge in some future generation, as a result of mechanisms such as haploid insufficiency, the expression of X-linked defects in male offspring or the future creation of double-recessive combinations. It also does not mean that we will not find defects if we look hard enough. The controversial discovery of fertility-threatening Y-chromosome deletions in the offspring of genotypically normal males as a consequence of ART, is an example of a condition that may take 25–30 years to surface even though the mutation was probably created shortly after fertilization [35].

Clearly, we must continue to be vigilant in our long-term monitoring of the health and wellbeing of children produced by ART. Given recent advances in our understanding of epigenetic defects in the spermatozoa of infertile male patients, we should also extend this surveillance to the DNA methylation profiles of children born as a result of assisted conception. It is also incumbent upon embryologists to optimize the quality of the gametes that are used for ART, particularly where ICSI is involved. The development of prophylactic antioxidant therapies [36], improved culture conditions [37], novel gamete selection technologies [38] and noninvasive methods for the assessment of embryo quality [39] will all contribute to the future evolution of ART as a safe, effective means of treating human infertility.

Financial & competing interests disclosure
Aitken is a Consultant for NuSep. The author has no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.

No writing assistance was utilized in the production of this manuscript.



References ChooseTop of pageINTRODUCTIONThe male factorParental ageEnvironmental pollutionErrors of endogenous meta...Electromagnetic radiation...Epigenetic damageIs ART a dangerous form o...References <<

Papers of special note have been highlighted as: • of interest •• of considerable interest

1 Van Voorhis‌ BJ. Clinical practice. in vitro fertilization. N. Engl. J. Med. 356, 379–386 (2007). [CrossRef] [Medline]
2 Adamson‌ GD, de Mouzon J, Lancaster P, Nygren KG, Sullivan E, Zegers-Hochschild F; International Committee for Monitoring Assisted Reproductive Technology. World collaborative report on in vitro fertilization, 2000. Fertil. Steril. 85, 1586–1622 (2006). [CrossRef] [Medline]
3 Hansen‌ M, Bower C, Milne E, de Klerk N, Kurinczuk JJ. Assisted reproductive technologies and the risk of birth defects – a systematic review. Hum. Reprod. 20, 328–338 (2005)
•• Meta-analysis suggesting a 30–40% increased risk of birth defects associated with assisted reproductive technology (ART).

[CrossRef] [Medline]
4 Jensen‌ TK, Sobotka T, Hansen MA, Pedersen AT, Lutz W, Skakkebæk NE. Declining trends in conception rates in recent birth cohorts of native Danish women: a possible role of deteriorating male reproductive health. Int. J. Androl. 31(2), 81–92 (2007).
• Recent review highlighting the declining fertility rates typical of the Danish population.

[CrossRef] [Medline]
5 Jorgensen‌ N, Carlsen E, Nermoen I et al. East–West gradient in semen quality in the Nordic–Baltic area: a study of men from the general population in Denmark, Norway, Estonia and Finland. Hum. Reprod. 17, 2199–2208 (2002). [CrossRef] [Medline]
6 Andersen‌ AN, Erb K. Register data on assisted reproductive technology (ART) in Europe including a detailed description of ART in Denmark. Int. J. Androl. 29, 12–16 (2006). [CrossRef]
7 McLachlan‌ RI, de Kretser DM. Male infertility: the case for continued research. Med. J. Aust. 174, 116–117 (2001). [Medline]
8 Aitken‌ RJ, De Iuliis GN. Origins and consequences of DNA damage in male germ cells. Reprod. Biomed. Online 14, 727–733 (2007)
•• Recent review of the causes and consequences of DNA damage in the male germ line.

[Medline]
9 Aitken‌ RJ, Koopman P, Lewis SE. Seeds of concern. Nature 432, 48–52 (2004).
• Review of potential environmental impacts on DNA damage in the germ line.

[CrossRef] [Medline]
10 Aitken‌ RJ, Krausz C. Oxidative stress, DNA damage and the Y chromosome. Reproduction 122, 497–506 (2001). [CrossRef] [Medline]
11 Fernández-Gonzalez‌ R, Moreira P, Pérez-Crespo M et al. Long-term effects of mouse intracytoplasmic sperm injection with DNA-fragmented sperm on health and behavior of adult offspring. Biol. Reprod. 78(4), 761–72 (2008).
•• Important recent paper providing experimental evidence that the performance of intracytoplasmic sperm injection (ICSI) with DNA-damaged spermatozoa can have long-lasting impacts on the health and wellbeing of the offspring.

[CrossRef] [Medline]
12 Irvine‌ DS, Twigg JP, Gordon EL, Fulton N, Milne PA, Aitken RJ. DNA integrity in human spermatozoa: relationships with semen quality. J. Androl. 21, 33–44 (2000). [Medline]
13 Schmid‌ TE, Eskenazi B, Baumgartner A et al. The effects of male age on sperm DNA damage in healthy non-smokers. Hum. Reprod. 22, 180–187 (2007). [CrossRef] [Medline]
14 Singh‌ NP, Muller CH, Berger RE. Effects of age on DNA double-strand breaks and apoptosis in human sperm. Fertil. Steril. 80, 1420–1430 (2003). [CrossRef] [Medline]
15 Crow‌ JF. The origins, patterns and implications of human spontaneous mutation. Nat. Rev. Genet. 1, 40–47 (2000). [CrossRef] [Medline]
16 Hamatani‌ T, Falco G, Carter MG et al. Age-associated alteration of gene expression patterns in mouse oocytes. Hum. Mol. Genet. 13, 2263–2278 (2004). [CrossRef] [Medline]
17 Yauk‌ C, Polyzos A, Rowan-Carroll A et al. Germ-line mutations, DNA damage, and global hypermethylation in mice exposed to particulate air pollution in an urban/industrial location. Proc. Natl Acad. Sci. USA 105, 605–610 (2008).
•• Important recent paper clearly demonstrating the impact that air pollution has on the epigenetic programming and integrity of sperm DNA.

[CrossRef] [Medline]
18 Rubes‌ J, Selevan SG, Evenson DP et al. Episodic air pollution is associated with increased DNA fragmentation in human sperm without other changes in semen quality. Hum. Reprod. 20, 2776–2783 (2005). [CrossRef] [Medline]
19 Rignell-Hydbom‌ A, Rylander L, Giwercman A et al. Exposure to PCBs and p,p´-DDE and human sperm chromatin integrity. Environ. Health Perspect. 113, 175–179 (2005). [Medline]
20 Meeker‌ JD, Singh NP, Ryan L et al. Urinary levels of insecticide metabolites and DNA damage in human sperm. Hum. Reprod. 19, 2573–2580 (2004). [CrossRef] [Medline]
21 Anderson‌ D, Schmid TE, Baumgartner A, Cemeli-Carratala E, Brinkworth MH, Wood JM. Oestrogenic compounds and oxidative stress (in human sperm and lymphocytes in the COMET assay). Mutat. Res. 544, 173–178 (2003). [CrossRef] [Medline]
22 Xu‌ DX, Shen HM, Zhu QX et al. The associations among semen quality, oxidative DNA damage in human spermatozoa and concentrations of cadmium, lead and selenium in seminal plasma. Mutat. Res. 534, 155–163 (2003). [Medline]
23 Rubes‌ J, Selevan SG, Sram RJ, Evenson DP, Perreault SD. GSTM1 genotype influences the susceptibility of men to sperm DNA damage associated with exposure to air pollution. Mutat. Res. 625, 20–28 (2007). [Medline]
24 Agbaje‌ IM, Rogers DA, McVicar CM et al. Insulin dependant diabetes mellitus: implications for male reproductive function. Hum. Reprod. 22, 1871–1877 (2007).
• Sentinel paper indicating that diabetic patients possess high levels of DNA damage in their spermatozoa.

[CrossRef] [Medline]
25 Shrilatha‌ B, Muralidhara. Early oxidative stress in testis and epididymal sperm in streptozotocin-induced diabetic mice: its progression and genotoxic consequences. Reprod. Toxicol. 23, 578–587 (2007). [CrossRef] [Medline]
26 Bennetts‌ LE, De Iuliis GN, Nixon B et al. Analysis of the impact of estrogenic compounds on DNA integrity in the male germ line. Mutat. Res. (2007) (In Press).
27 Wang‌ X, Sharma RK, Sikka SC, Thomas AJ Jr, Falcone T, Agarwal A. Oxidative stress is associated with increased apoptosis leading to spermatozoa DNA damage in patients with male factor infertility. Fertil. Steril. 80, 531–535 (2003). [CrossRef] [Medline]
28 Aitken‌ RJ, Baker HW. Seminal leukocytes: passengers, terrorists or good samaritans? Hum. Reprod. 10, 1736–1739 (1995).
•• Discussion of the significance of leukocytic infiltration in the origins of oxidative stress in the male reproductive tract.

[Medline]
29 Shaman‌ JA, Yamauchi Y, Ward WS. Sperm DNA fragmentation: awakening the sleeping genome. Biochem. Soc. Trans. 35, 626–628 (2007). [CrossRef] [Medline]
30 Banks‌ S, King SA, Irvine DS, Saunders PT. Impact of a mild scrotal heat stress on DNA integrity in murine spermatozoa. Reproduction 129, 505–514 (2005). [CrossRef] [Medline]
31 Carrell‌ DT. Paternal genetic and epigenetic influences on IVF outcome. Expert Rev. Obstet. Gynecol. 3(3), 359–367 (2008). [Abstract]
32 Houshdaran‌ S, Cortessis VK, Siegmund K, Yang A, Laird PW, Sokol RZ. Widespread epigenetic abnormalities suggest a broad DNA methylation erasure defect in abnormal human sperm. PLoS ONE 2, e1289 (2007). [CrossRef]
33 Anway‌ MD, Cupp AS, Uzumcu M, Skinner MK. Epigenetic transgenerational actions of endocrine disruptors and male fertility. Science 308, 1466–1469 (2005). [CrossRef] [Medline]
34 Gandini‌ L, Lombardo F, Paoli D et al. Full-term pregnancies achieved with ICSI despite high levels of sperm chromatin damage. Hum. Reprod. 19, 1409–1417 (2004). [CrossRef] [Medline]
35 Feng‌ C, Wang LQ, Dong MY, Huang HF. Assisted reproductive technology may increase clinical mutation detection in male offspring. Fertil. Steril. (2008) (Epub ahead of print).
• Recent publication indicating that the treatment of male infertility patients with ART is associated with the de novo appearance of Y chromosome deletions in the offspring.

36 Greco‌ E, Iacobelli M, Rienzi L, Ubaldi F, Ferrero S, Tesarik J. Reduction of the incidence of sperm DNA fragmentation by oral antioxidant treatment. J. Androl. 26, 349–353 (2005). [CrossRef] [Medline]
37 Friedler‌ S, Schachter M, Strassburger D, Esther K, Ron El R, Raziel A. A randomized clinical trial comparing recombinant hyaluronan/recombinant albumin versus human tubal fluid for cleavage stage embryo transfer in patients with multiple IVF-embryo transfer failure. Hum. Reprod. 22, 2444–2448 (2007). [CrossRef] [Medline]
38 Ainsworth‌ C, Nixon B, Jansen RP, Aitken RJ. First recorded pregnancy and normal birth after ICSI using electrophoretically isolated spermatozoa. Hum. Reprod. 22, 197–200 (2007). [CrossRef] [Medline]
39 Patrizio‌ P, Fragouli E, Bianchi V, Borini A, Wells D. Molecular methods for selection of the ideal oocyte. Reprod. Biomed. Online 15, 346–353 (2007). [Medline]
Website 101 Australian Babies. 4102.0. Australian Social Trends. Australian Bureau of Statistics (2007). www.abs.gov.au/AUSSTATS

Affiliations
R John Aitken
Laureate Professor of Biological Sciences, Faculty of Science and IT, University of Newcastle, Callaghan, NSW 2308, Australia. john.aitken@newcastle.edu.au