Abstract
Parenthood at an older age is becoming a trend among men, especially in the most developed societies. Aging has a significant impact on male fertility. Older men exhibit notable disturbances in the reproductive axis, with steroidogenesis being impacted much more than spermatogenesis. The endocrine changes, together with morphological and functional alternations of the aging testis, result in decreased testosterone production. Nonetheless, studies are needed to scrutinize the impact of age per se versus age-induced dysfunction of the reproductive axis. Furthermore, the multiple effects of aging on the acquisition of sperm motility, on sperm morphology and concentration indicate that the quality of spermatozoa declines over time, but few works have shed light on the molecular mechanisms that hamper sperm function in old men. In fact, this question is far from being completely answered and this is a subject of controversy. Hence, we will present an up-to-date review and discuss the molecular mechanisms involved in the alteration of the reproductive function in aging men. We will focus on the functioning of the reproductive axis and what are the major effects of aging in spermatogenesis. We will also discuss how aging affects sperm quality and possible causes underlying sperm dysfunction with special emphasis in oxidative stress.
Keywords: Aging, Men, Fertility, Spermatogenesis, Sperm quality, Oxidative stress.
Introduction
In recent years, there has been a tendency for parenthood among men of older ages. Statistics showed that in the last decade nearly two-thirds of babies have been conceived by fathers aged 30 and over [1]. In some countries, the mean age of fathers has increased from 29.2 years to 32.1 years. It seems that older fathers are no longer unusual, at least in modern societies. Unlike the abrupt decline in reproductive capacity observed in women, men maintain their reproductive function during life, though it gradually declines over time. Advanced paternal age is associated with changes in the production of reproductive hormones and sexual function. In men, the hypothalamus-pituitary testicular axis (HPT-axis) controls the release of sex hormones and ensures the formation and maturation of spermatozoa. Hormonal profile of sex steroids, and also spermatogenesis, suffer gradual changes induced by age- related alterations [2]. In fact, reproductive aging in men is characterized by a gradual decline in serum testosterone (T) levels from the fourth decade onwards [3]. Nevertheless, the exact mechanism for the decline of T levels remains to be fully elucidated. Alterations in the HPT- axis, decreasing numbers of Leydig cells (LCs), or both seem to be the major causes. LCs are
42 by luteinizing hormone (LH) secreted in pulses into the peripheral circulation by the pituitary gland, in response to gonadotropin-releasing hormone (GnRH) from the hypothalamus [4].
Alterations were reported in the steroidogenic pathway of aged males, which were associated with reduced T levels, including a decrease in LH secretion. Serum T levels decrease as a result of the reduced capacity of LCs to produce T [5, 6]. Additionally, older men also exhibit an altered pituitary secretion of follicle-stimulating hormone (FSH). Longitudinal studies with aging men have shown a rise in circulating levels of this gonadotropin [3, 7, 8]. Nevertheless, increased levels of FSH have been linked to impaired function of Sertoli cells (SCs), germ cell degeneration during meiosis and consequent impairment of spermatogenesis [9]. Male age is also associated with a reduced number of SCs and this is probably linked with a thickening of the basement membrane of seminiferous tubuli, in parallel with reduction of the seminiferous epithelium and reduced vascularization of the testes [10]. Altogether the alterations in HPT- axis and/or in testicular cells may have profound effects in the formation of sperm, a quantitative expression of spermatogenesis, which consists in the total number of spermatozoa produced per day [11].
Aging in males has been associated with altered sperm parameters [12-14]. In fact, several studies have showed that the quality of spermatozoa is not “immune” to the effects of age, though this is a subject of controversy. The different methodologies used in the analysis of sperm parameters and possible confounders, such as permanent exposure to environmental contaminants, metabolic diseases and factors associated to lifestyle, may explain the variability of the results [15-17]. In this context, it is imperative to shed light on the molecular foundations underlying the age-related changes of the reproductive parameters.
Spermatozoa are highly differentiated cells produced and released from seminiferous tubules and undergo a series of modifications in the epididymis, which are essential for the gradual differentiation that these cells suffer. Aging seems to be implicated in the alteration of some mechanisms involved in sperm formation, specially during the maturation process [18]. Little more information is available on how aging affects the quality of spermatozoa, particularly with respect to maturation-related events. In fact, this has been one of the greatest challenges for the reproductive biologists.
Along with the conventional causes for male infertility, oxidative stress has been considered one of the major causes for the age-associated sperm dysfunction. Oxidative stress occurs in aging, since as organisms age the efficacy of the mitochondrial function tends to diminish, due to impaired electron transport chain, thus increasing electron leakage. The relation between mitochondrial dysfunction and aging has also been long suspected, but dissecting its details remains to be fully elucidated. According with mitochondrial free radical theory of aging it has
43 of reactive oxygen species (ROS) [19]. ROS are oxidizing agents generated as byproducts from the metabolism of oxygen and include a range of radicals such as hydroxyl ion, superoxide ion and non-radicals such as hydrogen peroxide, among others [20]. A subclass of ROS has been described as reactive nitrogen species (RNS) and among them nitric oxide has been found to be crucial for various functions within the male reproductive system. It is generally accepted that excessive ROS production can be detrimental to sperm function and survival [21]. However, the available literature fails to show the exact levels of ROS that induce pathogenesis. Studies have shown that concentrations below 1 M play an important role in the regulation of a several signaling pathways [22]. Importantly, ROS-mediated signaling pathways are extremely complex partly due to the several biological effects induced by different levels of ROS. Currently, it is thought that low concentrations of ROS are essential for several sperm-related events. In fact, ROS regulate hyper activation, capacitation, acrosome reaction and consequently fertilization [23, 24]. Sperm incubated with low concentrations of hydrogen peroxide showed increased hyper activation, acrosome reaction and capacitation [21, 25]. On the other hand, the excessive production of ROS associated with aging, as well as the fact that the sperm are virtually devoid of antioxidant defenses, make these cells susceptible to ROS-induced damages. ROS promotes the oxidation of biomolecules, such as polyunsaturated fatty acids (PUFAs), which are the major components of the sperm plasma membrane. Lipid peroxidation promotes changes in membrane fluidity, causing the loss of motility and the consequent impairment of fertilization. On the other hand ROS are also responsible for DNA damage leading to its fragmentation [26]. Hence, oxidative stress has become a subject of great concern with several clinical studies, as well as studies using models of animal aging, indicating that there are significant changes that occur in spermatozoa as males enter advanced age and such changes have adverse reproductive outcomes, even for the progeny [27-30].
We will present an up-to-date review and discuss the molecular mechanisms involved in the alteration of the reproductive function in aging men. We will focus on the functioning HPT-axis and how spermatogenesis is governed with advancing age in males. We will also discuss the major effects of aging in sperm parameters and possible causes underlying sperm dysfunction, with special emphasis in oxidative stress.
The effects of aging in the male reproductive axis
Aging is a process that involves a considerable number of irreversible changes in all organs and systems, which are due to a variety of environmental and endogenous factors. In recent years, the effects of age on reproductive organs is becoming increasingly important, since a
44 suffer a drop in their reproductive capacities with advancing age, but in men this decline is less pronounced, allowing them to have children throughout life. The importance of women's age on the fertilization has been thoroughly studied and discussed in the literature. However, concerning paternal age there is still no consensus and this subject needs to be discussed in detail [31-34]. Aging induces profound alterations in the male reproductive system, namely in HPT axis, which consequently compromises testicular morphology and physiology. The reproductive axis consists of three essential parts: hypothalamus, anterior pituitary, and testes.
GnRH secreted from the hypothalamus reaches the anterior pituitary gland, via the hypophysial-portal system, stimulating the secretion of LH and FSH into the bloodstream by the gonadotropic cells. LH induces the production of T by the LCs, whereas FSH induces the SCs to secrete androgen-binding protein (ABP) and inhibin, playing also a key role in the initiation and progression of spermatogenesis [35]. The changes in the HPT axis with aging are complex and involve multiple levels. It is expected that aging causes a diminished secretion of GnRH, which in turn leads to reduced production of LH and consequently decreased T synthesis [36]. Still, in humans, direct measurement of GnRH is impossible due to its confinement to the hypophysial-portal circulation. As an alternative, the study of the LH pattern secretion has provided indirect evidence for a decrease in GnRH release with aging. Some aging men have showed lower LH pulse amplitude [37], while in others the pattern of LH release is completely chaotic [38]. The gonadotropin secretion and regulation in older men is complex and there is considerable conflicting data regarding the concentrations of gonadotropins, since there are studies reporting both hypogonadotropic (primary testicular disorder) and hypergonadotropic (secondary to hypothalamic-pituitary dysfunction) hypogonadism [39, 40]. For instance, Urban and collaborators [41] measured peripheric levels of LH and found no differences in the basal levels of LH between older and younger individuals.
In contrast, Zwart and collaborators [42] reported that gonadotropin responsiveness to GnRH was higher in older than in younger men. A possible explanation for the apparent dysregulation in the LH pulse amplitude in older men is that aging is associated with impaired pituitary responsiveness to GnRH. Additionally, studies with animal models have also shown a reduction in the LH release similar to that seen in humans [43]. Indeed, the number of synaptic inputs to GnRH neurons, as well as both GnRH transcritps and peptides, seems to decrease with age [44]. The Brown Norway rat has been widely used as a model of reproductive aging and has advantages over other strains, since these animals exhibit an unusual longevity without the confounder of illness. Some studies with this animal model have reported that the GnRH content of several hypothalamic areas is lower in older than in younger rats [45].
Moreover, older animals exhibit significant reductions in glutamate and gamma-aminobutyric
45 decreased hypothalamic excitatory amino acid expression and the reduced responsiveness of GnRH neurons may contribute to the altered LH pulsatile secretion observed in older rats [46].
Altogether, these disturbances in the hypothalamic-pituitary complex will certainly attenuate the testicular production of T [47, 48]. Most studies report an annual decrease of 0.5–1.5% of testicular T production, but the opinions about the clinical value of this modest decline differ [49]. T levels decrease with advancing age, not only due to disturbances in the hypothalamic- pituitary circuits, but also due to the impairment in the number of LCs and deterioration of testicular perfusion [5, 8, 50-52]. Gonads are not functional during the entire life of an individual and their normal function is not indispensable to the rest of the body. The endocrine functions of the testis begin in utero being interrupted between the neonatal period and puberty and continue thereafter with spermatogenesis, suffering only a slight decline with age [49]. One of the effects of the male reproductive system aging is an alteration of the testicular morphology during the different stages of lifespan [53]. Testicular aging is characterized by a large individual variability of changes that affect not only the testicular size, but also its morphology and function. Testicular volume increases between 30 and 60 years of age and gradually decreases after the 60 years of age [49, 53], probably due to HPT axis dysfunction. Mahmoud and collaborators correlated testicular volume measured by ultrasound with serum hormone levels in young and older males. Those authors found 31% decrease in testicular volume (21 ml versus 30 ml, respectively) in men with 75 years of age when compared with males between 18 and 40 years of age [54]. In a longitudinal study, testicular volume was evaluated in 150 men between the first month of age until 90 years. The mean testicular volume increased from birth, reaching the maximum volume at 25 years of age. After that, there was a slight but significant decline to a mean volume of 14 mL between 80 and 90 years of age [55]. This difference is related with meaningfully higher mean serum levels of gonadotropins and lower serum of free T [49, 54]. Furthermore, the testicular volume decrease in older men demonstrated a strong direct correlation with serum levels of inhibin B and inhibin B/FSH ratio, and indirect correlation with FSH. Age related changes in testes also include a decrease in the number of LCs, which act on feedback mechanisms and alter the pulsatile secretion of gonadotropins [55, 56]. Also, associations were found with T (direct) and LH (indirect) in old men and with the documented age-related decrease in the number of SCs [54, 57]. These cellular changes are accompanied by reduction in the capacity to secrete T and with elevated plasma levels of LH, FSH and 17β-estradiol.
Spermatogenesis in aging
Aging leads to changes in testicular cells that parallel the alterations seen in other parts of the body [58]. Male fertility is directly associated to the success of spermatogenesis, a continuous
46 production of spermatozoa. Spermatogenesis is a process regulated by several hormonal and endogenous factors [59-61] and occurs within the seminiferous tubules, the functional units of the testes, by a close cooperation between SCs and germ cells. During this process, immature germ cells undergo mitotic division, meiosis and differentiation to give rise to the millions of mature sperm cells [59, 62-64]. The testes maintain spermatogenesis throughout almost the entire adult life, notwithstanding some deterioration that might occur with aging [49]. In fact, with advancing age there is a decline of spermatogenic markers and, also a slight decrease of testicular gametogenic and endocrine function, with germ cells deterioration having a central role in the decline of the quantitative and qualitative aspects of spermatogenesis.
This impact of aging on fertility and spermatogenesis has been widely studied and a large body of evidence shows that paternal age exerts a negative impact on the quality of male germ cells [28, 29, 65-67]. Degenerative changes in the testes of older men have been described [2, 49, 68-70] and such changes include tubule involution, narrowing of tubular diameter, thickening of basal membrane associated with arrested spermatogenesis and fibrosis, and reduction of the mass of SCs and spermatogenic cells, with vacuolization of the former and multinucleation of the latter. SCs also accumulate lesions showing increased cytoplasmic lipid droplets, which may compromise spermatogenic efficiency [9]. Tubule involution is associated with an enlargement of the tunica propria, leading to progressive reduction of the seminiferous epithelium with progressive tubular sclerosis [71]. Aging in the human testes is also associated with reduction in the numbers and functional competence of LCs, non-Leydig interstitial cells and myoid cells [72]. Morphological characteristics of testes from aging men also include multilayered spermatogonia, reduced number of type-A dark spermatogonia, increased occurrence of multinucleated spermatogonia, as well as giant spermatids [71]. However, the onset and severity of testicular lesions may depend on individual variations [73]. Still, these changes tend to begin at the third decade of life and increase with age [74].
Advancing age is associated with a decline in semen quality and identifiable features include a decrease in the ejaculate volume, concentration of spermatozoa, and total sperm production as well as an increase in the percentage of nonviable and abnormal spermatozoa [74]. Studies on healthy older men have shown that aging is associated with a marked increase in the number of spermatozoa with abnormal flagellar midpieces, as well as a reduction in the percentage of motile spermatozoa [18]. Johnson and collaborators [57] reported a decline in the number of sperm per ejaculate as a function of age. According to these authors the number of spermatozoa per ejaculate is reduced by 30% by the seventh decade of life and by an additional 20% in the eighth decade. Nonetheless, the efforts to understand how sperm function is affected by age has been hampered by the difficulties on recruiting healthy
47 normalization of abstinence and sample selection. This often leads to inconsistent results.
Notwithstanding, it is generally accepted that sperm quality decreases as age advances [49].
Fertile individuals produce over an average of 40 million sperm per day, starting at puberty and expanding throughout all their reproductive life. valuation of quantitative aspects of spermatogenesis in aging men, expressed in terms of sperm production rates, is not a recent method and has showed that daily sperm production is significantly lower in older (50-80 years) than in younger (20-48 years) men with similar testicular weights [57] or lower testicular weights [69, 75].
The effects of aging in sperm quality
There is no defined age threshold regarding sperm production in men. However, male fertility seems to decline with aging, so it is important to know whether advanced paternal age is associated with diminished sperm quality and what are the major risks for male fertility in older individuals and to the offspring. Compelling evidence has shown that aging is associated with changes in sperm parameters [76-80]. Advancing aging is related to a decline in sperm parameters, specially sperm motility and morphology [28] (Table 1). Dondero and collaborators [78] reported a gradual decrease in sperm motility after 40 years of age. A negative correlation between advanced age and both sperm motility and morphology in healthy men was also confirmed by Centola and collaborators [77], however none of these studies adjusted for potential confounders, such as smoking and type of infertility among clinic patients. Auger and collaborators [76] compared semen between men in younger (30 years) and older (50 years) age groups. In this study, the authors adjusted data for duration of abstinence and observed that sperm motility and normal sperm morphology decreased by 0.6% and 0.9% each year, respectively. Other works using age as a categorical variable [80- 85] have shown a consistency toward decreasing percentage of sperm with normal morphology in older men. Indeed, the majority of the evidences available in literature seem to be coherent concerning the decrease in sperm motility and morphology with increasing age.
However, this consistency is not so obvious regarding sperm concentration (see Table 1).
Several studies have reported a decrease in sperm concentration with increased age [76-78, 80, 86]. Among these, it is interesting to note that Haidl and collaborators [80] found nearly half the sperm concentration in the older men (50 years) comparatively to young individuals (30 years). This is in line with Homonnai and collaborators [86] who found that the number of men with sperm concentration less than 5 million/mL was almost 3-fold higher in older men than in young men. Importantly, these results were observed in patients attending to infertility clinics, which may be a great contributor for such remarkable differences. Nevertheless, generalizing