Keywords

1 The Male Reproductive System

The male reproductive system consists of a number of sex organs that are part of the reproductive process. The testes are responsible for production of sperm and androgens, i.e. sex hormones essential to development and functional maintenance of the entire male reproductive tract. Each testis comprises two tissue compartments, which are functionally related but structurally separate: the seminiferous tubule compartment, lined with a complex epithelium of highly specialized Sertoli cells and developing spermatogenic cells, and the interstitial tissue compartment, which contains the androgen-producing Leydig cells, as well as the testicular vasculature, lymphatic and immune cells. The seminiferous tubules are connected through a structure called the rete testis, via a series of efferent ducts, to the adjacent epididymis, which concentrates and facilitates the maturation of the sperm. At ejaculation, epididymal fluid and sperm are propelled along the muscular vas deferens to the urethra, where they are combined with the secretions of the accessory glands to form the seminal plasma.

1.1 Spermatogenesis

Spermatogenesis is the process by which male spermatogonia develop into mature spermatozoa in sexually reproducing organisms. In mammals this process occurs in the male testes and epididymis in a stepwise fashion, and for humans takes approximately 64 days (Heller and Clermont 1963). Starting at puberty, it usually continues uninterrupted until death, although a slight decrease can be discerned in the quantity of the sperm produced with increase in age. The entire process can be broken up into several distinct stages, i.e. spermatocytogenesis, spermatidogenesis, and spermiogenesis. The initial stages occur within the testes and progress to the epididymis, where the developing gametes mature and are stored until ejaculation. The seminiferous tubules of the testes are the starting point for the process, where stem cells adjacent to the inner tubule wall divide in a centripetal direction to produce immature sperm. Maturation occurs in the epididymis and involves the acquisition of a tail and hence motility.

In spermatocytogenesis, a diploid spermatogonium in the basal compartment of seminiferous tubules divides mitotically to produce two diploid intermediate cells called primary spermatocytes.

On the basis of the appearance of the nuclei, three functionally separate spermatogonial cell types are recognized: type A dark spermatogonia , type A pale spermatogonia, and type B spermatogonia. The population of spermatogonia is maintained by type A dark spermatogonia, which do not directly participate in producing sperm and simply ensure a supply of stem cells. Type A pale spermatogonia repeatedly divide mitotically to produce identical cell clones. When repeated division ceases, the cells differentiate into type B spermatogonia. This stage is referred to as the spermatogonial phase. Type B spermatogonia undergo mitosis to produce diploid primary spermatocytes.

Each primary spermatocyte then moves into the adluminal compartment of the seminiferous tubules, duplicates its DNA and subsequently undergoes meiosis I to produce two secondary spermatocytes. This division implicates sources of genetic variation, such as random inclusion of either parental chromosome, and chromosomal crossover, to increase the genetic variability of the gamete.

Secondary spermatocytes rapidly enter meiosis II and divide to produce haploid spermatids in spermatidogenesis. Owing to the brevity of this stage, secondary spermatocytes are rarely seen in histological preparations. Each cell division from a spermatogonium to a spermatid is incomplete; the cells remain connected to one another by bridges of cytoplasm to allow synchronous development.

During spermiogenesis, the spermatids begin to grow a tail, and develop a thickened mid-piece, where the mitochondria gather and form an axoneme. Spermatid DNA also undergoes packaging, becoming highly condensed. The DNA is packaged firstly with specific nuclear basic proteins, which are subsequently replaced with protamines during spermatid elongation. The resultant tightly packed chromatin is transcriptionally inactive. The Golgi apparatus surrounds the now condensed nucleus, becoming the acrosome. One of the centrioles of the cell elongates to become the tail of the sperm . Maturation then takes place under the influence of testosterone , which is involved in the removal of the remaining unnecessary cytoplasm and organelles. The excess cytoplasm, known as residual bodies, is phagocytosed by surrounding Sertoli cells in the testes. The resulting spermatozoa are now mature but lack motility, rendering them sterile. The mature spermatozoa are released from the protective Sertoli cells into the lumen of the seminiferous tubule in a process called spermiation. The non-motile spermatozoa are transported to the epididymis in testicular fluid secreted by the Sertoli cells by peristaltic contraction. While residing in the epididymis, they acquire motility and become capable of fertilization. However, transport of the mature spermatozoa through the remainder of the male reproductive system is achieved via muscle contraction rather than the spermatozoon’s recently acquired motility. During spermatogenesis, Sertoli cells support the developing gamete by maintaining the environment necessary for development and maturation via the blood–testis barrier, secreting the substances initiating meiosis, the androgen-binding protein, and inhibin, by phagocytosing residual cytoplasm left over from spermiogenesis (Fig. 1).

Fig. 1
figure 1

Spermatogenesis and site of action of methylxanthines

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1.1.1 Methylxanthines and Spermatogenesis

Studies on the teratogenic or sperm -injuring potential of methylxanthine started nearly 40 years ago by investigating the effects of caffeine on spermatogenesis (Ax et al. 1976). It is to note that all animal studies have demonstrated that, depending on the method of administration and the species, the developmental no-observed-effect level (NOEL) is approximately 30 mg/kg per day, the teratogenic NOEL is 100 mg/kg per day, and the reproductive NOEL approximately 80–120 mg/kg per day (Christian and Brent 2001). Roosters, fed 0.1% caffeine mixed by weight into a standard ration (about 145 mg/day), after 14 days of treatment showed a significant decrease in fertility . Semen output and sperm concentration were markedly reduced after 17–21 days of treatment, and no semen could be collected from the roosters after a 30-day treatment. Testicular histological investigation showed interruption of spermatocyte divisions and abnormal spermiogenesis, but removal of dietary caffeine resulted in resumption of semen production and a return of fertility to the control level. In rats fed caffeine, theobromine , or theophylline at a very high dietary level of 0.5% by weight into a standard ration (LD50 = 200 mg/kg, Fredholm et al. 1999) for periods ranging from 14 to 75 weeks, a significant positive finding was the occurrence of severe bilateral testicular atrophy with aspermatogenesis or oligospermatogenesis in 85–100% of the rats (Weinberger et al. 1978; Friedman et al. 1979). The relative testicular toxicity of the methylxanthines was reported as caffeine being the most potent, theobromine slightly less potent, and theophylline considerably less potent. Somewhat variable atrophic changes of the accessory sexual organs (epididymis, prostate, and seminal vesicles) accompanied the testicular changes. Cytogenetic analysis of testes from caffeine- or theophylline-fed rats revealed a significantly reduced number of mitotic cells in the caffeine-treated group. Plasma testosterone concentrations were significantly elevated in the theobromine group and were elevated in the caffeine-treated group; this correlated morphologically with an apparent hyperplasia of interstitial cells in severely atrophied testes in these groups. Plasma cholesterol concentrations were significantly increased in the caffeine and theobromine groups (Gans 1982; Ettlin et al. 1986; Ezzat and El-Gohary 1994; Funabashi et al. 2000). Studies of the toxicities of theobromine and cocoa extract on the reproductive tract of male rats showed that theobromine and high-dose cocoa extract caused vacuolation within the Sertoli cell, abnormally shaped spermatids , and failed release of late spermatids in treated animals. However, the frequencies of some parameters of testis alterations were significantly lower in the high-dose cocoa-extract-treated group compared with the theobromine-treated group, demonstrating the ability of a cocoa extract containing theobromine to alter testis structure in a similar pattern but with reduced intensity compared with that observed after oral exposure to pure theobromine (Wang et al. 1992; Wang and Waller 1994).

The effects of caffeine at a concentration of 0.5% and fed to male rats for 7 weeks were compared with those of 0.8% dietary theobromine . Both dietary methylated xanthines produced significant decreases in food consumption and body-weight gain in rats when compared with their respective control groups. The theobromine-fed rats showed severe testicular atrophy with extensive spermatogenic cell degeneration and necrosis, while the testes of rats fed caffeine showed only scattered vacuolar degeneration of spermatogenic cells. Caffeine appeared to be more potent than theobromine as an anorexic agent in rats, but to be equivalent to theobromine in its potential for inducing thymic atrophy and spermatogenic cell destruction with testicular atrophy (Gans 1984). Long-term intake of caffeine caused suppression of spermatogenesis mainly through inhibition of the release of follicle-stimulating hormone (FSH). Daily administration of caffeine (30 or 60 mg/kg) to mature male rabbits for four consecutive weeks caused an increase in the plasma FSH level and a decrease in the luteinizing hormone (LH) level. A light microscope study revealed reduced size of the seminiferous tubules, inhibition of spermatogenesis, fatty degeneration of the liver, and hepatic lesions, whereas the adrenal glands exhibited signs of stimulated steroidogenesis (Ezzat and El-Gohary 1994). Caffeine , when administered to the rat (30 mg/kg/day) during pregnancy, affected certain aspects of normal sexual differentiation of the fetal gonads (Pollard et al. 1990). In the male fetus, caffeine significantly inhibited differentiation of the interstitial tissue and Leydig cells, with a significant consequent reduction in testosterone biosynthesis in the fetal testes. Caffeine also had an effect on the earlier morphogenic organization of the seminiferous cords. In the female fetus, caffeine did not modify ovarian differentiation nor the morphology of the ovaries, tissue arrangement, and overall appearance. These results indicating that caffeine, when administered during pregnancy, significantly inhibited the differentiation of the seminiferous cords and subsequent Leydig cell development in the interstitium, prompted the investigation of whether the observed effects were caused either by direct effects of caffeine or by intermediary secondary toxic effects of metabolites, i.e. theophylline and theobromine . Explants of 13-day-old fetal testis were cultured for 4 days in vitro in the presence of graded doses of caffeine, theophylline, or theobromine. Fetal testes exposed to caffeine or theobromine differentiated normally, developing seminiferous cords made up of Sertoli and germ cells, soon followed by the differentiation of functionally active Leydig cells appearing in the newly formed interstitium. In contrast, explants exposed to theophylline failed to develop seminiferous cords and, as a consequence, Leydig cells (Pollard et al. 2001). Recently, theophylline was shown to induce infertility by causing germ cell apoptosis in the testicular seminiferous epithelium. Theophylline exposure altered the expression of the genes within the ubiquitin–proteasome pathway (UPP), implicated in spermatogenesis and epididymal sperm quality control (Tengowski et al. 2007). Results suggest that the reprotoxic exposure alters the tissue-specific expression of UPP genes in the testis and epididymis, which may contribute to the aberrant spermatogenesis and epididymal processing of both normal and defective spermatozoa. Moreover, theophylline induced infertility by incapacitating the nurturing Sertoli cells, thus resulting in the premature release of late-differentiating spermatogenic cells, round spermatids (Weinberger et al. 1978). This leads to the depletion of spermatids and mature spermatozoa from the adluminal compartment of the seminiferous epithelium, ultimately causing testicular atrophy (Strandgaard and Miller 1998;Tengowski et al. 2005).

Other authors have suggest a beneficial effect of caffeine in the regulation of male gamete maturation since, acting as an agonist of ryanodine receptors, which induce release of Ca2+ from intracellular stores in spermatogonia , pachytene spermatocytes, and round spermatids , caffeine modulates calcium mobilization and plays a fundamental role in spermatozoa maturation (Chiarella et al. 2004). Conflicting results still seem to characterize the association between male caffeine consumption in adult life and semen quality, whereas the association between prenatal coffee consumption and semen quality and levels of reproductive hormones seems to be responsible for a small to moderate effect on semen volume and the levels of reproductive hormones (Ramlau-Hansen et al. 2008).

1.2 Acquisition of Sperm Fertility

Mammalian spermatozoa emerging from the male reproductive tract are incapable of fertilizing eggs. They acquire this ability either during transit in the female reproductive tract (Yanagimachi 1994) or during incubation in suitable in vitro media (Allegrucci et al. 2001). Such conditioning, called capacitation , renders the spermatozoa capable interacting with the oocyte and thereby inducing the acrosome reaction (AR ). Capacitation and AR are related to many effectors and signal transduction pathways, but the molecular basis of these processes is still to be fully elucidated (Kopf and Wilde 1990; Florman et al. 1992; de Lamirande et al. 1997; Tulsiani et al. 1998, 2007; Thundathil et al. 2002; Morales et al. 2007; Salicioni et al. 2007; Aitken et al. 2007; Wassarman 2009; Abou-haila and Tulsiani 2009). Sperm capacitation is a multistep process that involves several biochemical and ultrastructural changes in the sperm membrane, ranging from modification of membrane lipid composition to an increased permeability to ions. The efflux of membrane cholesterol leads to bovine sperm capacitation (Visconti et al. 1999). Albumin, high-density lipoproteins, and follicular and oviductal lipoproteins are capacitation effectors of human and bovine spermatozoa (Moreau et al. 1998; Thérien et al. 2001). Capacitation is correlated with an increase of protein tyrosine phosphorylation (Visconti et al. 1995a, b, 1999; Aitken et al. 1998) and the signal transduction pathway leading to protein tyrosine phosphorylation is thought to be central to either the attainment of the capacitative state (Visconti and Kopf 1998) or the concomitant expression of hyperactivated motility (Mahony and Gwathmey 1999; Si and Okuno 1999). As capacitation proceeds, several proteins undergo serine/threonine phosphorylation or threonine/tyrosine double phosphorylation (Thundathil et al. 2002). The AR is an exocytotic process by which lytic enzymes are released from the sperm acrosome and digest the zona pellucida so that spermatozoa can reach and fertilize the oocyte (Yanagimachi 1994; de Lamirande et al. 1997). Sperm AR occurs within minutes, cannot be reversed once it is induced, and can be triggered in vitro by different inducers, such as zona pellucida (Yanagimachi 1994), progesterone (Harrison et al. 2000), calcium ionophores, lysophosphatidylcholine (de Lamirande et al. 1997), follicular fluid (De Jonge et al. 1993), and ATP (Luria et al. 2002). Sperm AR takes place after fusion between the acrosome and the overlying plasma membrane and involves calcium influx, actin polymerization, a rise in intracellular pH, and protein activation (phospholipases, kinases, G proteins, etc.) (Yanagimachi 1994; Baldi et al. 2000; Liguori et al. 2005; Abou-Haila and Tulsiani 2009).

1.2.1 Methylxanthines and Acquisition of Sperm Fertility

The greatest part of the data in the literature reports the use xanthines/methylxanthines as phosphodiesterase (PDE) inhibitors that maintain intracellular levels of cyclic AMP (cAMP) , thereby acting as motility-enhancing agents or capacitating effectors (Hong et al. 1981; Jiang et al. 1984; Depeiges and Dacheux 1985; Galantino-Homer et al. 1997; Leclerc et al. 1998; Jaiswal and Majumder 1998; Harayama et al. 1998; Leclerc and Goupil 2002; Buffone et al. 2005; Lachance et al. 2007; Yeste et al. 2008).

In a study finalized to clarify the role of the adenosine A1 receptor in the acquisition of fertilizing capacity, caffeine was used as an adenosine A1 receptor antagonist at low concentrations that binds to and inhibits half of the adenosine receptors (Fredholm et al, 1999). This dose of caffeine had very little, if any, effect on the acquisition of the capacitated status, whereas a very high dose of caffeine caused a significant reduction in sperm capacitation (Minelli et al. 2004) (Fig. 2). However, it is of note that high concentrations of caffeine are unlikely to be reached by caffeine consumers since strong side effects would preclude ingestion of this amount. Hence, the data suggest that regular caffeine consumption is unlikely to significantly affect spermatozoa function, thereby reassuring all coffee drinkers of the lack of negative effects of caffeine on male fertility . The scientific literature dealing with the AR contains references to the use of methylxanthines as PDE inhibitors (Kopf et al. 1983a, b; Carr and Acott 1990; Ain et al. 1999; Lachance et al. 2007) that maintain intracellular cAMP levels and induce the AR.

Fig. 2
figure 2

a Percentage of capacitated cells as function of incubation time in A1R+/+ (filled circle), A1R+/− (filled triangle), and A1R−/− (filled square) mouse spermatozoa (filled square). b Percentage of capacitated cells as function of incubation time of A1R+/+ murine sperm in the presence of 15 μM caffeine (filled triangle) and 100 μM caffeine. (Adapted from Minelli et al. 2004)

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2 The Female Reproductive Tract

The female reproductive tract is a dynamic system cycling under the control of the key ovarian steroid hormones oestrogen and progesterone. It contains the uterus, which act as the receptacle for male sperm , and the ovaries, which produce the egg cells. The Fallopian tubes attach the uterus to the ovaries, which, at certain intervals, release an ovum , which passes through the Fallopian tube into the uterus. If, in this transit, the ovum meets with sperm , the sperm penetrate and merge with the egg , fertilizing it. The diploid zygote then implants itself in the wall of the uterus, where it begins the processes of embryogenesis and morphogenesis.

2.1 Oocyte Maturation

Despite the universal requirement of a haploid gamete for sexual reproduction, meiosis is regulated differently in oocytes and spermatocytes. As shown in Fig. 1, spermatocytes proceed through the meiotic divisions uninterrupted, whereas in female mammals, meiosis occurs over a prolonged period of time (Eppig et al. 2004). Mammalian oocytes are engaged in a complex meiotic cell division, characterized by several “stops and starts”, and after resuming meiosis, they rely on maternal factors to sustain the subsequent developmental steps until the maternal-to-zygotic transition occurs. The process by which the oocyte completes the first meiotic division and undergoes other cytoplasmic changes and progresses to metaphase II is called oocyte maturation (Fig. 3). Because the mature, fertilizable oocyte has a relatively short lifespan in the female reproductive tract, the timing of oocyte meiotic arrest, as well as maturation, is tightly regulated (Mehlmann 2005). The functional unit within the ovary is the follicle, formed during embryonic development, and it comprises one or more layers of granulosa cells surrounding the oocyte (Gougeon 1996; Zeleznik 2004). During follicular growth, the somatic cells divide to form several layers, the oocyte enlarges, and a fluid-filled antrum begins to form. Some follicles at the early antral stage are “recruited” to continue growing; this growth is dependent on the pituitary gonadotropin FSH (Gougeon 1996; Zeleznik 2004). During this phase, the antrum divides the granulosa cells into two separate compartments: mural granulosa cells form the outer layers, while the cumulus cells surround the oocyte . The oocyte grows to its full size (about 75-μm diameter in the mouse, about 100 μm in the human), but remains arrested in prophase I. If an oocyte is removed from an antral follicle, it spontaneously resumes meiosis and progresses to second metaphase (Pincus and Enzmann 1935). This indicates that the follicle cells hold the oocyte in prophase arrest. Meiosis resumes in response to a surge of LH from the pituitary gland during the oestrous or menstrual cycle, shortly before ovulation. LH receptors (LHRs) are located on the mural granulosa cells but not on the cumulus cells or the oocyte (Peng et al. 1991; Richards et al. 2002), so the mechanism(s) by which LH stimulates oocyte maturation is indirect. Prior to the midcycle surge of LH, the growing oocyte acquires the ability to undergo oocyte maturation. The acquisition of meiotic competence occurs around the time of antrum formation (Mehlmann et al. 2004) and corresponds to a point at which the oocyte achieves a threshold level of maturation-promoting proteins, such as cyclin-dependant kinase (Cdk1) and cyclin B (Kanatsu-Shinohara et al. 2000). Meiotic arrest is regulated by cAMP levels within the oocyte (Conti et al. 2002; Eppig et al. 2004) since the cAMP level affects the activity of the Cdk/cyclin B protein complex, also known as maturation/meiosis or mitosis promoting factor (MPF). High cAMP levels result in the phosphorylation of Cdk1 on Thr14 and Tyr15, rendering it inactive (Duckworth et al. 2002), while a decrease in cAMP levels leads to the dephosphorylation of Cdk1 on Thr14 and Tyr15, the MPF complex becomes active, and the oocyte can re-enter meiosis. A hypothesis for how high levels of cAMP are maintained in competent, fully grown oocytes is that the oocyte produces its own cAMP through a G-protein-linked receptor in the oocyte plasma membrane that stimulates Gs and, subsequently, adenylyl cyclase (AC). Direct evidence for an essential role of Gs in the maintenance of meiotic arrest was obtained by microinjecting either a function-blocking antibody or a dominant negative form of the α-subunit of Gs into follicle-enclosed oocytes (Mehlmann et al. 2002; Kalinowski et al. 2003). This pathway was confirmed by the finding that oocytes from mice lacking the AC3 AC isoform, which is present in the oocyte , spontaneously undergo germinal vesicle breakdown within ovarian follicles (Horner et al. 2003).

Fig. 3
figure 3

Oogenesis and site of action of methylxanthines

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Recently, it was shown that heat shock transcription factor 1, which triggers the transcription of several genes encoding heat shock proteins, is highly expressed in oocytes and plays an important role in normal progression of meiosis by directly regulating Hsp90α expression (Metchat et al. 2009).

2.1.1 Methylxanthines and Oocyte Maturation

As for male gametes, the largest part of the data in the literature reports the use xanthines/methylxanthines as PDE inhibitors that maintain the intracellular levels of cAMP responsible for the meiotic arrest. Several groups (Cho et al. 1974; Dekel and Beers 1978; Schultz et al. 1983a, b; Vivarelli et al. 1983; Bornslaeger and Schultz 1985; Törnell et al. 1990; Haider and Chaube 1996; Webb et al. 2000; Conti et al. 2002) showed that spontaneous maturation of oocytes isolated from their follicles can be prevented by including membrane-permeant cAMP analogues or cAMP PDE inhibitors, such as hypoxanthine and 3-isobutyl-1-methylxanthine (IBMX), in the culture medium. The cAMP levels decrease in oocytes following their removal from their follicles as well as in isolated oocytes after removal of IBMX. The decrease in the level of oocyte cAMP occurs within 2 h after washing out IBMX, a time during which the oocyte becomes committed to resuming meiosis and cAMP levels increase in isolated oocytes. When transported into oocytes from the cumulus cells via gap junctions, cAMP also plays an important role in the regulation of meiotic progression beyond the meiosis I (MI) stage (Shimada et al. 2002). Using selective PDE inhibitors, such as milrinone (a PDE3 inhibitor), cilostamide (a PDE3 inhibitor), and rolipram (a PDE4 inhibitor), studies focused on the differential regulation of cAMP levels within the oocyte and somatic (cumulus) cell compartments of the follicle showed that specific PDE subtypes are differentially localized within the two compartments of the follicle, i.e. the type 3 PDE in the oocyte and the type 4 PDE in the granulosa cells (Thomas et al. 2002). Moreover, oocyte cAMP levels are primarily regulated in oocytes by its degradation by PDE, whereas granulosa cell cAMP levels are controlled mainly by active AC, with both sources able to participate in oocyte meiotic regulation. IBMX does not interfere with the expression of LHR in cumulus cells surrounding oocytes, whereas the binding of LH to its receptor induces a further increase in cAMP level, progesterone production, and acceleration of meiotic progression to the metaphase I stage. The role of cAMP in the oocyte meiotic arrest was further supported by Laforest et al. (2005), who showed a fundamental significance of cAMP pathways in controlling meiotic resumption in porcine oocytes. This control is at two levels, the ability to synthesize cAMP via active AC, where the cyclase of porcine oocyte is sensitive to forskolin, and the degradation of cAMP via cilostamide-sensitive PDE. A more detailed study of the effects of IBMX on the oocyte meiotic block (Barretto et al. 2007) showed that IBMX is able to prevent resumption of meiosis by maintaining elevated cAMP concentrations in the oocyte, whereas roscovitine, a purine known to specifically inhibit MPF kinase activity, maintains bovine oocytes at the germinal vesicle stage, indicating that the meiotic inhibitors delay the progression of nuclear maturation without affecting cytoplasmic maturation. It was proposed that the inhibitory cAMP is synthesized within oocytes via a stimulatory α-subunit of G protein. After the presence of Gs-α molecules in porcine oocytes had been shown, an anti-Gs-α antibody was injected into porcine immature oocytes and this inhibition of ooplasmic Gs-α functions significantly promoted germinal vesicle breakdown of the oocytes, whose spontaneous meiotic resumption was prevented by IBMX treatment. Moreover, although cyclin B synthesis and MPF activation were largely prevented until 30 h of culture in IBMX-treated oocytes, injection of anti-Gs-α antibody into these oocytes partially recovered cyclin B synthesis and activated MPF activity at 30 h, suggesting that meiotic resumption of porcine oocytes is prevented by ooplasmic Gs-α, which may stimulate cAMP synthesis within porcine oocytes, and that synthesized cAMP prevents meiotic resumption of oocytes through the signalling pathways involved in MPF activation (Morikawa et al. 2007). More recently, Ozawa et al. (2008) focused their attention on cAMP content, gap-junctional communication status, and LHR expression in porcine cumulus–oocyte complexes treated with IBMX or with FSH. They found that the inhibition of PDEs in porcine cumulus–oocyte complexes makes the oocyte ready for release from meiotic arrest, whereas the maintenance of a moderate cAMP content may prolong gap-junctional communications and stimulate LHR expression. A recent paper (Pirino et al. 2009) showed that meiotic resumption requires activation of the MPF. Protein kinase A (PKA) activity sustains the prophase arrest by inhibiting Cdk1. Therefore, the inhibition of the activity of the Cdc25 protein required for MPF activation results in mitotic arrest. Phosphorylation of a highly conserved serine 321 residue of Cdc25B 21 plays a key role in the negative regulation and localization of Cdc25B during prophase arrest, suggesting that Cdc25B is a direct target of PKA.

2.2 Caffeine and Female Fertility

Caffeine is a subject of interest among consumers and health professionals because it is widely consumed in the diet by most segments of the population and can exert several pharmacological effects (Dews 1982; Fredholm 1995; Christian and Brent 2001; Mandel 2002; Derbyshire and Abdula 2008; Yu et al. 2009).

The medical literature contains many varied references indicating that human adverse reproductive/developmental effects are produced by caffeine. Although it is difficult to compare doses of caffeine in animals and humans, the medical literature dealing with developmental and reproductive risks of caffeine underwent a thorough revision by evaluating the biological plausibility of the epidemiological and animal findings. When comparing effects among different species, one can only accomplish dose equivalence be by considering the results of pharmacokinetics studies, metabolic studies, and dose-response investigations in the human and the species being studied. Moreover, the importance of dose within a species is of fundamental concern when determining developmental risks since most drugs/chemicals are potentially associated with developmental toxicity/teratogenicity only at some exposure level. The genetic constitution of an organism, i.e. both the maternal and the fetal genotypes, is also an important factor in the susceptibility of a species. Indeed, more than 30 disorders of increased sensitivity to drug toxicity or effects have been reported in the human owing to an inherited trait (McKusick 1988). Unlike human epidemiology studies, which are difficult to control and with multiple inherent flaws that prevent identification of causality, animal studies are conducted under conditions in which all the variables can be better controlled. In addition, current non-clinical studies generally include identification of achieved blood levels/exposures in the maternal animal and the developing offspring, a critical factor because the severity of the effect is related to the ability of the conceptus to recover from the insult (Johnson and Christian 1984). Nevertheless, results of non-clinical animal studies provide excellent tools for predicting potential effects of caffeine on human reproduction and development. Indeed the LD50 of caffeine is fairly consistent across species, including Homo sapiens (Dews, 1982). The plasma level resulting from 1.1 mg/kg caffeine (a single cup of coffee containing 80 mg of caffeine ingested by a 70-kg human) ranges from 0.5 to 1.5 mg/L. A similar dose–concentration relationship is found in many species, including rodents and primates (Hirsh 1984). It is generally assumed that 10 mg/kg in a rat represents about 250 mg of caffeine in a human weighing 70 kg (3.5 mg/kg), and that this would correspond to about two to three cups of coffee.

Nawrot et al. (2003) reviewed the effects of caffeine on human health and concluded that for the healthy adult population, moderate daily caffeine consumption at levels up to 400–450 mg/day (5.7–6.4 mg/kg/day in a 70-kg adult, equivalent to four to five cups per day) was not associated with adverse effects, which include general toxicity, effects on bone status and calcium balance, cardiovascular effects, behavioural changes, increased incidence of cancer, and effects on male fertility . However, the authors also reported that children and women of reproductive age were “at risk” subgroups who might require dietary advice to moderate their caffeine intake. High levels of caffeine intake may delay conception among fertile women (Bolúmar et al. 1997). The effects of caffeine consumption on delayed conception were evaluated in a European multicentre study on risk factors of infertility in a randomly selected sample of 3,187 women aged 25–44 years. A significantly increased odds ratio for subfecundity in the first pregnancy was observed for women drinking more than 500 mg of caffeine per day (more than six cups), the effect being relatively stronger in smokers than in non-smokers. Women with the highest level of consumption had an increase in the time leading to the first pregnancy. In addition, women whose caffeine consumption was high had less than a third of the risk for a long menses (8 days or more) compared with women who did not consume caffeine. Those whose caffeine consumption was high also had a doubled risk for a short cycle length (24 day or less); this association was also evident in those whose caffeine consumption was high but did not smoke. However, caffeine intake was not strongly related to an increased risk for anovulation, short luteal phase (10 days or less), long follicular phase (24 days or more), long cycle (36 days or more), or measures of within-woman cycle variability (Fenster et al. 1999). The mean birth weight was reduced by high reported caffeine consumption, but this small decrease in birth weight, observed for maternal caffeine consumption, is unlikely to be clinically important except for women consuming 600 mg of caffeine daily (more than 7.2 cups) (Bracken et al. 2003). More recently, in a study finalized to determine whether smoking, alcohol, and caffeine may be related to the four indicators of ovarian age, i.e. antral follicle count, FSH, inhibin B, and oestradiol, and therefore to fecundability and fertility (Kinney et al. 2007), 188 women, aged 22–49, were investigated and least-squares was regression used to estimate differences in antral follicle count and hormone levels for women who smoke cigarettes or who drink alcohol or caffeine. Current smoking is related to elevated FSH levels, but not to the antral follicle count, inhibin B, or oestradiol. Neither alcohol nor caffeine was found to be related to any ovarian age indicator, suggesting that caffeine, at the dosage of 156 mg/day (1.9 cups), does not affect ovarian age indicators. On the basis of data from a large retrospective epidemiology study and from a large retrospective case-control study in humans, it appears that use of caffeine does not impair ovulation to the point of decreasing fertility and during pregnancy has little, if any, effect on the outcome of pregnancy. Nevertheless, although caffeine use during pregnancy does not appear to be associated with substantial risk and the association between soft drinks and ovulatory disorder infertility does not seem to be attributable to their caffeine content, most clinicians recommend that pregnant women limit their consumption of foods, beverages, and drugs containing caffeine, since caffeine crosses the placenta (Care Study Group 2008; Chavarro et al. 2009). Recent research (Björklund et al. 2008) has confirmed the concerns about caffeine consumption during pregnancy or the early postnatal period because there may be long-lasting behavioural changes after caffeine exposure early in life. Indeed, pregnant wild-type mice, given modest doses of caffeine (0.3 g/L in drinking water), gave birth to offspring that as adults exhibited increased locomotor activity in an open field. The offspring also responded to cocaine challenge with greater locomotor activity than mice not perinatally exposed to caffeine. The same behavioural experiments on mice heterozygous for adenosine A1 receptor gene, where signalling via adenosine A1 receptors is reduced to about the same degree as after modest consumption of caffeine, showed a behavioural profile similar to that of wild-type mice perinatally exposed to caffeine. It appeared that the mother’s genotype was critical for behavioural changes in adult offspring, suggesting that perinatal caffeine, by acting on adenosine A1 receptors in the mother, causes long-lasting behavioural changes in the offspring that even manifest themselves in the second generation. Mice homozygous for genetic deletion of the adenosine A1 receptor showed a reduction in number of offspring and increased time between litters (Table 1) (Minelli et al. 2004). Interestingly, caffeine and some of its derivatives present in coffee leaves affect egg -laying by the coffee leaf miner Leucoptera coffeella, one of the main coffee pests in the Neotropical region. In fact, increased leaf levels of caffeine favour egg-laying by the coffee leaf miner with a significant concentration–response relationship, providing support for the hypothesis that caffeine stimulates egg-laying by the coffee leaf miner in coffee leaves (Magalhães et al. 2008).

Table 1 Effects of male genotype
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3 Assisted Reproductive Techniques

Since the birth of the first baby conceived with in vitro fertilization (IVF) and embryo transfer, assisted reproductive technology, an extremely successful form of therapy for many infertile couples, is currently practised all over the world.

3.1 Methylxanthines in Assisted Reproductive Techniques

The effects of AC, IBMX, and dibutyryl cAMP (dbcAMP) on porcine oocyte in vitro maturation, IVF, and subsequent embryonic development were investigated by Somfai et al. (2003). They showed that a change in the intracellular level of cAMP during oocyte collection does not affect the maturational and developmental competence of the oocytes and that synchronization of meiotic maturation using dbcAMP enhances the meiotic potential of oocytes by promoting the MI to metaphase II transition and results in high developmental competence by monospermic fertilization. In IVF experiments, IBMX in association with FSH and LH, was used to synchronize the oocytes. At 6 days after IVF, the blastocyst rate in oocytes matured under these conditions was significantly higher than that for oocytes cultured in the absence of LH. The results suggest that the treatment of oocytes with FSH and IBMX causes the expression of LHR in cumulus cells, holds the oocytes at the germinal vesicle II stage, and can be considered as a beneficial procedure to obtain in-vitro-matured oocytes with high developmental competence (Shimada et al. 2003a, b). Besides PKA, several protein kinases are involved in oocyte maturation, and studies of the mechanisms of protein kinase B (PKB) activation and its role in cumulus cells during in vitro meiotic resumption of oocytes showed that the addition of PDE inhibitors maintained the level of PKB activity in cumulus cells at levels comparable with those in cumulus cells just after collection from their follicles and that the inhibitory effect of hypoxanthine on spontaneous meiotic resumption was overcome by addition of a PKB inhibitor.

3.2 Caffeine in Assisted Reproductive Techniques

Recently, Maalouf et al. (2009) reported the effects of cumulus cell removal and caffeine treatment on the development of in-vitro-matured ovine oocytes. Whereas removal of cumulus cells and aging increases polyspermy, caffeine was effective in reducing this phenomenon, showing that caffeine treatment statistically increases the development to blastocyst and lowers the frequency of polyspermy.

Caffeine increases MPF and mitogen-activated protein kinase activities in ovine oocytes, prevents age-related changes, and increases cell numbers in blastocysts produced by somatic cell nuclear transfer (Lee and Campbell 2006, 2008). Used in experiments of nuclear remodelling of somatic cell nuclear transfer embryos and subsequent development and DNA methylation patterns, caffeine induces premature chromosome condensation at a high rate, a high blastocyst formation rate, and lowers the apoptotic cell index, suggesting that the nuclear remodelling type controlled by caffeine treatment can affect in vitro development and the methylation status of nuclear transfer in relation to nuclear reprogramming (Kwon et al. 2008). These results confirmed previous observations that showed that caffeine treatment promotes nuclear remodelling although it does not prevent the decrease in the developmental ability of cloned embryos caused by oocyte aging (Iwamoto et al. 2005; Kawahara et al. 2005). On the other hand, with use of a mouse model, it was shown that caffeine had no effect on the quality of oocytes matured in vivo, whereas it was detrimental to the quality of oocytes matured in vitro (Miao et al. 2007). However, in vivo studies with female mice administered 150 mg/kg caffeine at various times prior to metaphase I, showed non-significant differences in the frequencies of hyperploid, MI, diploid, premature centromere separation, single chromatids, and structural chromosome aberrations between the controls and each of the caffeine groups (Mailhes et al. 1996; Jaakma et al. 1997).

Studies of the effects of caffeine on male gametes showed stimulation of sperm capacitation and spontaneous AR (Funahashi 2003, 2005); therefore, during IVF procedures, supplementation with β-mercaptoethanol, which neutralizes the stimulatory effect of caffeine, has a beneficial effect in maintaining the function of gametes, the incidence of normal fertilization, and, consequently, the quality of IVF embryos. However, a limited exposure of gametes to caffeine significantly reduced the mean number of sperm cells that penetrated into the oocyte and asynchrony in the morphology of sperm nuclei in polyspermic oocytes (Funahashi and Romar 2004). Other epidemiology studies evaluated the timing and amount of caffeine intake by women and men undergoing IVF and gamete intra-Fallopian transfer (GIFT) on oocyte retrieval, sperm parameters, fertilization, multiple gestations, miscarriage, and live births. A prospective study of 221 couples was conducted between 1993 and 1998. “Usual” caffeine intake during the lifetime and 1 year prior to the study, caffeine intake during the week of the initial clinic visit, as well as caffeine intake during the week of the procedure were evaluated for beverages (coffee, soda, and tea) and chocolates. Not achieving a live birth was significantly associated with “usual” female caffeine consumption for an intake of more than 50 mg/day and consumption of 0–2 mg/day during the week of the initial visit. Infant gestational age decreased by 3.8 or 3.5 weeks for women who consumed more than 50 mg/day of caffeine “usually” or during the week of the initial visit. The odds of having multiple gestations increased by 2.2 and 3.0 for men who increased their “usual” intake or intake during the week of the initial visit by an extra 100 mg/day. Caffeine intake was not significantly associated with other outcomes. This was the first IVF/GIFT study to report any effect of caffeine on live births, gestational age, and multiple gestations and if these findings are replicated, caffeine use should be minimized prior to and while undergoing IVF/GIFT (Klonoff-Cohen et al. 2002).

4 Conclusion

Reports on methylxanthines and their effects on reproduction have mainly focused on their use as in vitro PDE inhibitors that, by maintaining high intracellular levels of cAMP , differently affect female and male gametes. Animal studies have largely shown that methylxanthines have toxic effects on gonads and gametogenesis of both sexes, although comparing doses of caffeine in animals and humans is for many reasons not an easy task. Caffeine is present in many beverages (coffee, tea, colas, and chocolate) and in over-the-counter medications. The medical literature contains many varied references that appear to indicate that human adverse reproductive/developmental effects are produced by caffeine. However, if caffeine causes such effects, the reproductive consequences could be very serious because caffeine-containing foods and beverages are consumed by most of the human populations of the world, and, as world “coffee culture” continues to grow, world caffeine intakes continue to increase. After revising the medical literature dealing with developmental and reproductive risks of caffeine on the basis of the biological plausibility of the epidemiological and animal findings and the methods and conclusions of previous investigators, clinical counsellors can inform prepregnant/pregnant women who do not smoke or drink alcohol and who consume moderate amounts of caffeine (5–6 mg/kg per day, five cups) that they do not have an increase in reproductive risks or adverse effects.