Several studies have shown a dramatic reduction of semen quality in many industrialized countries and infertility is becoming a public health top priority, whose incidence is associated to late-onset adult diseases, especially cancer, shorter life expectancy and trans-generational effects. The male reproductive system is particularly sensitive to a broad variety of reproductive and developmental toxicants, including many environmental pollutants and recent studies suggest that human semen is an early and sensitive environmental and health marker. A set of semen biomarkers is described for reproductive health effects in relation to environmental exposure, where human semen seems to be an early and sensitive source of biomarkers than blood to monitor high environmental pressure on human health. Environmental health should consider reproductive health and development, from intrauterine life to childhood and puberty: these are both vulnerable targets and high-value protection goals, inasmuch as they represent the future of our societies. Hence, biomarkers of reproductive health should be exploited as early signals of environmental pressure and increased risk of adverse chronic health effects so that the use of “human seminal model” might be the main objective to be considered in the agenda of public prevention policies for early detection and innovative programs of health surveillance in environmental risk areas.
- semen quality
- DNA sperm damage
- environmental marker
- health marker
- endocrine disruptors
- sperm telomere
- redox status
- reproductive health
- environmental health
Since the early 1950s, in several demographic surveys a steady decline of birth rates in all European countries has been observed . In particular semen quality was highly decreased in many industrialized countries [2, 3, 4] and in many European, Japanese and American young people poor semen quality was associated with subfertility or even infertility [5, 6]. The risk is that semen quality of a significant proportion of young men in developed countries will impair the fecundity potential causing on a short-term basis just a longer waiting time to pregnancy without to considerably family sizes of modern couples [7, 8], but on a middle-, long-term basis, strongly contributing (along with socio-economic factors) to the already observed European decrease in the birth rate. While there was a considerable variability in trends in sperm counts over the past 20 years, several recent studies have reported that 20–30% of young men today have sperm concentration below 40 × 106/ml, which is associated with reduced fecundity [9, 10, 11]. Among life-style changes that contribute to a reduced birth rate, affecting semen parameters and/or semen quality, there are: increased age at conception of both parents (although as a consequence of socio-economic factors), the increase in obesity, physical inactivity and the exposure to environmental and dietary environmental and chemical contaminants, including drugs. Exposure to man-made chemicals, in particular in the workplace, is recognized as major risk factors for male infertility in both epidemiological and experimental studies [12, 13, 14, 15, 16]. Individuals exposed for professional reasons to environmental contaminants show a reduction of concentration, motility, morphology and/or sperm DNA damage. In addition, toxicological studies in animal models are reporting DNA damages or epigenetic alterations within the germline: exposure to environmental xenobiotics during the fetal development and in early post-natal life, caused congenital malformations or reproductive tissue alterations or reduced fertility or signs of reproductive syndromes, such as the testicular dysgenesis syndrome, in particular when multiple
With the release of the Silent Spring in 1962  the issues related to chemical pollution have begun to become a topic of political and scientific debate by laying the basis of environmental chemistry and ecotoxicology as we know them. Environmental toxicology concerns the way in which toxic substances reach the organism and affect human health. At present many chemicals  have been detected in tissues and biological fluids of human body (Figures 1 and 2).
2.1. Organic pollutants and reproduction
Persistent organic pollutants (POPs) are very durable toxic chemicals which include polychlorinated dibenzodioxins polychlorinated dibenzofurans polychlorinated biphenyls (PCBs), chlorinated organic pesticides, PAHs, hexachlorobenzene and many other substances that we find in daily life such as polybrominated diphenyl ethers (PBDEs), perfluorooctane sulfonate, Perfluorottanoic acid ammonium salt, brominated flame retardants, food additives such as bisphenols and phthalates (plasticizers) and parabens (preservatives), according to recent experimental acquisitions, are known as endocrine disruptors (Endocrine Disrupting Chemicals). They are able to interfere with the production, release, transport, metabolism, binding, action or elimination of natural hormones of the body responsible for maintaining the homeostasis and the setting of endocrine reproductive processes [57, 58, 59]. They can also alter the cellular oxido-reductive homeostasis (redox status), resulting in a condition known as biochemical oxidative stress [60, 61, 62] a genotoxic action featuring a genetic and epigenetic damage transmissible through the germ line to the offspring (transgenerational effect). This last aspect is definitely very disturbing to future generations’ public health and justifies the growing interest of the scientific community in the study of the reproductive system in recent years [63, 64, 65]. These substances, very stable and soluble in fats, are found in semen that has a considerable lipid amount [66, 67].
2.2. Inorganic pollutants and reproduction
Metals toxicity depends on several factors, including their ability to bonds to reactive groups of enzymes and proteins (e.g. thiol groups) thus altering their structure and/or function. They may also interfere with the bioaccumulation of essential metals (e.g. iron, calcium and zinc) thus negatively affect those physiological mechanisms depending upon their bioavailability. Heavy metals accumulation in living organisms, in particular lead, cadmium, arsenic, mercury, depend upon the exposure to contaminated environment and may trigger acute and chronic degenerative diseases: In particular, genotoxic elements (Arsenic, Cadmium and Nickel) may damage the DNA structure either directly (through the production of oxygen radicals) or indirectly (via the alteration of enzymes responsible for DNA repair) and they may interfere in the activities of regulators of proliferation, apoptosis, differentiation and cell transformation [68, 69, 70, 71]. Metals also include “trace metals,” such as zinc, copper, iron, manganese, present in humans under physiological conditions, which are toxic at high concentrations. The risk assessment of the exposure to metals is achieved through human biomonitoring studies and their quantification in human biological fluids such as blood, serum and urine, being an indispensable tool to evaluate the possible influence of environmental determinants on human health. The level of metals in human fluids reflects the amount entering the body
2.3. Mechanisms involved in male reproductive dysfunction
2.3.1. Oxidative stress
Oxidative stress plays an important role in the etiology of male infertility by impairing negatively the quality and the function of the sperm  although the relationship between the bioaccumulation of environmental pollutants and the alteration of the seminal redox status has not been elucidated yet, and neither the possible mechanism of action. The imbalance of antioxidant defenses and detoxification processes provides a logical explanation to the onset of diseases caused by oxidative stress in men  and increases the organism susceptibility to pollutants toxicity . After all, the balance between oxidation and anti-oxidation is critically important in maintaining healthy any biological system.
The fact remains however, that pro-oxidant activity of PM  PAHs  on human health has been demonstrated in clinical data, whereas the harmful effects caused by toxic heavy metals or pesticides organophosphates  have been proved in animal studies. Reactive oxygen species (ROS), at low physiological levels, play an important role in sperm maturation and function . On the contrary, excessive amounts of ROS produced by leukocytes and immature spermatozoa can damage mature sperm and DNA integrity [82, 83, 84]. The mechanism of DNA damage by ROS is mainly due to the high susceptibility of spermatozoa to ROS for their high content of polyunsaturated fatty acids, major components of cellular and intracellular membranes (Figure 3). An increase in oxidative stress has been found in 80% of infertile men clinically tested, and it seems that exposure to environmental toxicants contributes to this increment [60, 78, 79, 80]. In addition, a positive correlation between ROS and sperm DNA fragmentation has been reported in studies . However, the Nuclear factor (erythroid-derived 2)-like 2 (Nrf2), plays a key role in the modulation of antioxidant response, which basically modulates both synthesis and the recycling of the main cellular antioxidant, that is the reduced glutathione. The reduced activity of glutathione reductase, has been associated with oxidative stress-related diseases , just like an increased susceptibility to adverse effects induced by pollutants  has also been associated with increased expression of p53  (Figure 4).
Notably, detoxifying/antioxidant defenses can be modulated by diet. However, it is known that improper eating habits (
2.3.2. Genetic alterations
Endocrine Disrupting Chemicals affect spermatogenesis both through alterations in the hypothalamic–pituitary axis, and direct damage to spermatozoa [90, 91, 92]. In recent decades, several studies have shown disorders of spermatogenesis due to genetic causes (15–30% of infertile males) [93, 94] and chromosomal aberrations, either numerical or structural, can profoundly affect fertility. It is estimated that the frequency of chromosomal aberrations in the general population is about 0.6% , and 2–14% in infertility male . In particular, chromosomal aberrations increase with the increasing severity of infertility. Moreover, some genetic polymorphisms involved in the metabolism and detoxification activities as well as in DNA repair capacity influence individual susceptibility to environmental exposure leading to changes in sperm quality . The main alteration responsible for male infertility is represented by DNA and chromatin alterations, highly sensitive to exogenous contaminants . Some studies have suggested that environmental toxins affect sperm DNA’s integrity and it has been observed that exposure to air pollutants such as PM, is capable of producing disomy of sexual chromosome in nemasperm DNA . In fact, most chromosomal abnormalities are lethal and so they either manifest as a sperm’s inability or as miscarriage .
In particular, aneuploidy defined as structural and numerical aberrations of chromosomes , is an informative effect biomarker, for male reproductive toxicants and a hallmark of cancer [101, 102, 103, 104]. There are some substances known that induce sperm aneuploidy and can be carcinogenic [105, 106] and for this reason sperm aneuploidy is associated with both increased risk of cancer and reproductive toxicity. Fortunately, sperm aneuploidy assessment has become very easy and this opens up to a growing use of health risk assessment from chemical hazard  so that, it could be integrated with current aneuploidy and chromosome imbalance assessments in place for somatic cells . In conclusion, sperm aneuploidy evaluation is informative well beyond the standard sperm parameters (number, motility, morphology) useful for comprehensive evaluation of carcinogenicity and reproductive toxicity.
With regard to sperm DNA’s integrity, it is a great indicator of male fertility, since men with normal sperm parameters may also have a high degree of DNA fragmentation, leading cause of undiagnosed /inexplicable infertility. In fact, the damage to sperm DNA contributes not only to infertility, but also on the frequency of miscarriages and birth defects in the offspring. The data supporting this are primarily derived from animal toxicology studies, which unequivocally demonstrate that the genetic integrity of the male germ line play an important role in determining the normal embryonic development .
The results of studies by toxicologists using several compounds in increasing doses prove adverse effects on the development of the embryo, on animal behavior, postnatal growth, longevity of progeny, as well as increased susceptibility to cancer. These toxicology animal-data support the hypothesis that toxic substances can act on the male germ line by interfering with the development of human pregnancies and the health of the unborn. To support this, there are associations between paternal smoking, oxidative DNA damage of sperm and the incidence of cancer in children. The origins of sperm DNA damage are not yet clearly defined, but in light of recent discoveries, six main mechanisms are hypothesized: (1) apoptosis during the process of spermatogenesis; (2) breakage of DNA strands created from the sperm chromatin remodeling during the process of spermatogenesis; (3) post-testicular DNA fragmentation induced mainly by oxygen radicals, including nitric oxide and hydroxyl radicals, during the transport of spermatozoa through the seminiferous tubules and epididymis; (4) DNA fragmentation induced by endogenous caspase and endonuclease; (5) DNA damage induced by radiotherapy and chemotherapy; (6) DNA damage induced by environmental toxins . The damage in testicular sperm DNA is statistically lower than what is found in ejaculated sperm . Sperm nuclear DNA fragmentation is the last phase of apoptosis, a highly controlled programmed cell death program that plays a key role in different biological processes such as embryonic development and maintenance of homeostasis. High cell proliferation rate and cell differentiation processes occur during maturation from stem cell to haploid mature sperm. Apoptosis is needed to avoid the excess of cell proliferation and it seems to have a role in germ cells differentiation. This process might also be induced by several environmental stimuli or damages . In case of DNA damage within the male germ line, the adverse outcome(s) will depend either from the type of damage or from the genomic region affected or from the timing of the damage itself and, as an overall consequence, from the ability of the embryo repair system to properly counteract any damage earlier than the first mitotic division will occur. In any case, the embryo could not always effectively repair damages carried on from male germ as it occurs in genetic dominant diseases, such as achondroplasia . Furthermore, healthy children born with assisted reproduction from DNA-damaged sperm  may possess genetic or epigenetic alterations generating a phenotypic change in the next generation(s) due to double recessive gene expression or in the birth of a male upon chromosome X mutations. Finally, it is also possible that DNA-damaged sperm can cause offspring defects not recognized at birth. The recent discovery that DNA damage in sperm of males due to aging is associated with the onset of epilepsy, schizophrenia, autism, and bipolar illness [115, 116].
Strikingly, an increased risk of sperm DNA fragmentation was associated to high levels of air pollution, in fact seems that the classical sperm parameters -motility, concentration, morphology- do not change related to high smog levels, while sperm DNA fragmentation appeared to be much more sensible . In this direction, also in Campania Region (Southern Italy), preliminary data of EcoFoodFertility initiative , indicated an increased sperm DNA damage associated to environmental pressure, measured with two techniques. In fact, healthy, no-smoking, no-drinker, no professionally exposed to environmental stresses males (n = 175, mean age 30 ± 4) were enrolled in areas of High or Low environmental impact. According to their stable residence in “Land of Fires”, a wide area between the towns of Naples and Caserta (High Environmental Impact Area—HIP; n = 70) or in Alto-Medio Sele in Salerno province (Low Environmental Impact Area – LIP; n = 105), data of the enrolled men were compared by their DNA Fragmentation Index (DFI). DFI was evaluated by using the sperm DNA fragmentation Kit (Halosperm®, Halotech DNA SL). Furthermore, the spermatic p53 levels were also assessed by using the DuoSet® ELISA (R&D) . The results obtained so far support the effectiveness of the considered markers in the quantification of DNA damages as well as the relationship between the extent of the observed sperm DNA damage and the environmental characteristics of the area of residence (HIP versus LIP areas). In conclusion, these data showed sperm DNA damage measured as DFI by SCD and p53 overexpression to be an early and sensitive marker of environmental pollution .
In recent years, an increasing interest has been directed on other biomarkers of DNA integrity in male germinal cells: telomere. Telomeres are noncoding double-stranded DNA repeats (in humans, TTAGGG sequences extended 10–15 kbIn dividing cells, the synthesis of new telomeric DNA repeats requires the activity of telomerase, a protein complex composed of the TERT enzyme and of the telomere-associated proteins, able to recognize the 150–200 nt 3′-single stranded (G-strand) overhang. During aging in most adult somatic cells, a progressive telomere shortening occurs and, in turn, telomerase activity decrease or completely disappear. In contrast to such adult somatic cells, germ cells maintain high telomerase activity, long telomeres and high proliferative potential [119, 120]. In particular, the sperm telomere length (STL) seems to be of fundament importance for fertilization and early embryo development . To date, the relationship between telomere function and aspects of semen quality is an area of great attention. Indeed, it has been reported that sperm TL is lower in oligozoospermic than in normozoospermic men . Furthermore, spermatozoa from elderly males have significantly longer telomeres than those from younger males, but the biological implications of this paradoxical effect are unknown . Additionally, telomere dysfunction is a relevant mechanism driving cancers in humans . Indeed, critical telomere attrition results in chromosomal aberration which in the absence of normal cellular DNA repair and apoptosis can lead to genetic instability. On the other hand, long telomeres may permit cells to escape growth arrest and increase the chance of acquiring mutations, especially in the presence of an external exposure, i.e. smoking and sun exposure. In fact, longer telomeres have been associated with some types of cancers, especially melanoma and lung cancer . Recently, a Mendelian randomization study reported that longer telomeres were associated with increased risk of several cancers but reduced risk of some non-neoplastic diseases .
Interestingly, accumulating evidence indicates that leukocyte telomeric DNA may be one important target of environmental [127, 128, 129, 130, 131, 132]. Accordingly, a very recent study has shown a possible association between high environmental pressure in polluted area and the STL . In particular, a preliminary study was carried out evaluate the influence of environmental exposure to the telomere length (TL) of leukocytes (LTL) and of STL. This pilot study was conducted on young healthy men living in HEI or in LEI area and the data obtained showed that STL was significantly greater in subjects while no significant difference was observed between LTL and HEI in the LEI group and no correlation between STL and sperm parameters was found . These findings support the view that STL is a more sensible marker than LTL to environmental pollution and it is a further evidence that the genetic structure of spermatozoa is particularly sensitive to environmental insults.
2.3.3. Epigenetic alterations
In recent years, interest has grown on new acquisitions that regulate gene expression and epigenetic mechanisms. In fact, if the interaction between genes and environment in determining human phenotypes has been known for many years, the real innovation provided by epigenetic studies concerns specific gene expression changes without any change in their sequence. Therefore, as genetic variants make the organism vulnerable to certain environmental insults, epigenetic alterations induced by the environment may have the same effect and especially could be transmitted to the offspring. Thus, birth defects, greater susceptibility to diseases in adulthood, may be the result of a gene/environment interaction that occurred in one of the parents, not the subject itself. Studying the sperm epigenome represents a new frontier in the field of human reproduction, and numerous studies have shown the importance of epigenetic mechanisms as potential biomarkers in hazard identification and risk assessment attributable to environmental exposures. Epigenetic mechanisms responsible for these alterations are represented by DNA methylation, histone modifications and noncoding microRNAs . The association between sperm DNA methylation and idiopathic male infertility is already documented with studies [136, 137, 138, 139]. Other studies have shown that DNA hypermethylation of gene promoters (like
2.4. The semen as an early marker of environmental exposure (environmental sentinel)
Semen qualitative and quantitative changes observed by several epidemiological studies, by Carlsen and latest ones [2, 3, 4, 5], show how these changes are induced by individual lifestyle and from the environment. Epidemiological studies on individuals exposed for professional reasons or living in contaminated areas and nearby settlements, demonstrate significant alterations of the semen: reduction of the motility, concentration, of sperm’s morphology, sperm DNA damage, sperm aneuploidies, alteration of sperm epigenome that result in increased cases of infertility, recurrent miscarriage, congenital malformations. Toxicological studies conducted on mice, show how some of the major environmental organic and inorganic contaminants reduce seminal quality. Significant changes of semen quality are noticed in different environments [22, 23, 24, 25, 26, 27]. Exposure to air pollution has been associated with abnormalities in sperm parameters. In recent studies the negative effect on sperm motility was estimated, in particular on sperm DNA’s integrity from carbon monoxide, nitrogen dioxide, sulfur dioxide, ozone, lead and PM 2.5, the latter being of particular interest, because it contains several trace elements and PAHs, powerful endocrine disruptors .
Spermatogenesis unlike oogenesis from puberty onward is continuously and therefore more easily exposed to insults in his stages of continuous replication. Moreover, biologically a 20-year-old’s sperm has undergone about 160 rounds of chromosome replication, a 40-year-old’s has undergone 610 and many male germline mutations fall into the “replicative” category or the “non-replicative,” such as those caused by environmental exposure, so male germline accumulates mutations faster than female one [48, 49]. For instance, it is thought that sperm cells are more susceptible than eggs to the effects of oxidative damage as a consequence of: (i) the limited cytoplasmic space where to host the enzymes involved in the antioxidant protection, and (ii) the higher amount of polyunsaturated fatty acids within the sperm membranes rendering them more susceptible to oxidative stress, such as lipid peroxidation . Furthermore, in semen it is possible to measure simultaneously environmental contaminants and
2.5. The semen as an early marker of health (health sentinel)
The spermatogenesis cycle is extremely complex and vulnerable to endogenous and exogenous stress, so it is not surprising that it can be an important indicator of the state of well-being of the organism. Recent studies have demonstrated the association between semen quality and state of health, correlating the semen quality with either chronic degenerative diseases, comorbidities and even mortality [36, 42, 43, 51, 52, 53].
In a first study of Eisenberg  a group of 9387 men was examined, average age 38 years, which had been evaluated for infertility issues between 1994 and 2011. Within the group, 44% had at least one medical diagnosis not related to infertility. Using the Charlson Comorbidity Index, researchers have shown that men with a higher index of chronic conditions had a lower count of sperm volume and motility, of total number of sperms and of normal shape. Sperm abnormalities rates were significantly higher among men with endocrine-metabolic, circulatory or genitourinary disorders and skin diseases, compared to other men without these conditions. Vascular hypertension, cerebrovascular disease and ischemic heart disease were associated with higher rates of sperm abnormalities. On the other hand, about 15% of all human genes are directly involved in reproduction and the majority of these genes may also play an important role in other parts of the body.
In a second study of Eisenberg  2238 men recruited in an infertility clinic of Texas were analyzed: 451 of which with azoospermia and 1787. It was compared the incidence of cancer on with that on the general population of Texas. At the first evaluation of infertility, the average age was 35.7 years. After a 6–7 years follow-up, it was shown that 29 of the infertile men developed a cancer, 10 (2.2%) among those ones with azoospermia and 19 (1.1%) among those ones without it. In comparison to the overall population of Texas, this subset of infertile men had a significantly higher risk of overall cancers and such a was significantly higher in men with azoospermia than in those without azoospermia.
The same Eisenberg linked semen quality with mortality rates  and found that men with damaged seminal parameters, including low sperm volume, concentration, sperm motility, had higher death rates than men with normal sperm parameters. Men with at least two abnormal sperm parameters had a 2.3-fold higher death risk (95% CI 1.12–4.65) than men with normal sperm. This further study of association, shows that men with poor semen parameters have an increased mortality rate in subsequent years and suggests that the fertility assessment may be an indicator of overall health.
A certain number of regions in all the world experience a higher incidence of health disorders (reproductive, pediatric, cancer, etc.) due to environmental pollution: the societal costs associated with poor health and the interventions to reduce pollution are stirring debates and concerns. It is important a science-based guidance for preventing/reducing health risks in many high environmental pressure areas.
Information about levels of exposure to contaminants (chemical, physical) is critical to evaluate and to manage environmental and professional risks and, as a result, as much as possible, to measure the biological risk expressed in terms of probability of reaching potential harm through the exposure to certain chemical and/or physical stress. There are new analytical tools today that first identify and measure biomarkers, quantitative end-point and intermediate pathways of biological tissue/fluid fluids to identify early signs of functional or structural modification before clinical damage. Therefore, in order to have greater preventive efficacy and raise the level of attention and protection especially to populations living in areas with greater environmental exposure, it is important consider to organofunctional “
The authors would like to thank EcoFoodFertility research group and Silvia Letizia Piscopo for the English revision.