Selenium

Selenium is generally prescribed for ageing. There are 25 selenoproteins in the human (male), among which the most well known are the glutathione peroxidases (GPX). For male fertility, the only reason for which selenium is prescribed, without restriction as a supplement, is its role as a co-factor for phospholipid hydroperoxide glutathione peroxidase (GPX 4, PHGPx, Ursini et al.,Reference Ursini, Maiorino and Roveri1997), which is responsible for the compaction of the spermatozoon nucleus (Fig. 2), and in fact, of the tertiary structure of the nucleus. This enzyme allows protamines at the cystine bridges to padlock the DNA. Several observations argue against the uncontrolled use of selenium supplementation in male infertility.

Figure 2 Antioxidants at too high levels block this process (importance of glutathione).

First, there is little or no selenium deficiency described in the scientific literature for the ‘Caucasian’ population. The usefulness of supplementation can therefore be questioned. Optimum concentrations range between 50 and 70 μg/ml. Within and beyond these values, the quality of the spermatozoon is altered (Bleau et al., Reference Bleau, Lemarbre, Faucher, Roberts and Chapdelaine1984). Second, even if selenium supplementation increases serum values it does not modify testicular values – as shown in animal studies (Hawkes et al., Reference Hawkes, Alkan and Wong2009; Combs et al., Reference Combs, Watts, Jackson, Johnson, Zeng, Scheett, Uthus, Schomburg, Hoeg, Hoefig, Davis and Milner2011). Third, together with aluminum, bacterial contamination, radioactivity, pesticides and nitrates, a high concentration of selenium is one of the six parameters used to regulate safety in human drinking water, according to European Economic Community (EEC) regulations.

In addition, other basic elements should be considered: selenites prompt apoptosis by generating superoxides (oxygenated free radicals) via the mitochondria (Xiang et al., Reference Xiang, Zhao and Zhong2009), which is the primary source for DNA damage in the spermatozoon due to its metabolism in relation to its energy-supplying (ox-phos) role (Koppers et al., Reference Koppers, De Iuliis, Finnie, McLaughlin and Aitken2008). In DNA repair processes, interaction may occur between the ‘zinc finger’ structure as well as damage from selenium and other elements, whose presence is essential although in trace form. These interactions have anti-carcinogenetic effects when the elements are present in weak concentrations and compromise genetic stability at strong concentration levels by affecting the methylation of DNA (Davis et al., Reference Davis, Uthus and Finley2000; Davis & Uthus, Reference Davis and Uthus2003; Hartwig et al., Reference Hartwig, Blessing, Schwerdtle and Walter2003). In a study that involved 100 patients, Li et al. (Reference Li, Zhong, Jiang, Wang, Zuo and Sha2012) found that the Cu, Mn, and Se concentrations were significantly higher in seminal plasma of an ‘abnormal sperm group’ than ‘the normal sperm group’ (P (Cu) = 0.024, P (Mn) = 0.002, P (Se) = 0.002). According to Hawkes & Turek (Reference Hawkes and Turek2001), selenium at high doses reduces sperm motility significantly through a modification of thyroid hormone metabolism. Finally, the concentration of selenium in serum is not a marker of oxidative stress, except in the case of rheumatoid type diseases (Brack et al., Reference Brack, Brack, Bonnefont-Rousselot, Dreyfus, Chapman and Kontush2012), in which it actually increases.

Co-enzyme Q10 (coQ10)

First, it is important to note that coQ10 deficiency is rare and occurs exceptionally and primarily in the case of recessive autosomal mutations, of diagnosed cancer or treatments with statins (known to induce DNA fragmentation in sperm). CoQ10 deficiencies are also present in case of neurovegetative disorders such as diabetes, muscular and cardiovascular pathologies (Villalba et al., Reference Villalba, Parrado, Santos-Gonzalez and Alcain2010). Generally speaking, coQ10 deficiency would not be expected to be a significant problem in the relatively young population exposed to ART. The second important fact is that commercially available products of coQ10 (ubiquinone) are absorbed poorly by the intestine, primarily due to its very low solubility (Liu & Artmann Reference Liu and Artmann2009; Villalba et al., Reference Villalba, Parrado, Santos-Gonzalez and Alcain2010). Therefore, the bioavailability appears to be low or close to zero. This observation is confirmed by Nadjarzadeh et al. (Reference Nadjarzadeh, Sadeghi, Amirjannati, Vafa, Motevalian, Gohari, Akhondi, Yavari and Shidfar2011) – in a double-blinded randomized study using coQ10 against a placebo there is no improvement in the quality of the spermatozoon. Marginal increases in coQ10 seminal plasma concentration may be observed with long-term (6-month) treatments, but with no improvement in fertility (Mancini & Balercia, Reference Mancini and Balercia2011) In fact, only recently with ubiquinol, a reduced form of coQ10, protection against peroxidation of the membrane lipids has been observed, which may explain a partial improvement of sperm mobility among certain patients. In other patients, this type of prolonged treatment inhibits the production of spermatozoa (reduced to 10% of the original count; T. Amar, personal communication).

Vitamin C

It is well known that vitamin C can have a pro-oxidant role in the presence of divalent cations such as iron and copper (and Se?). The most serious problem is the negative role of excessive amounts in terms of compaction, i.e. the tertiary structure of the spermatozoon nucleus. It is important to note that the quality of the tertiary structure is absolutely essential for the success of the initial cell cycles and early embryonic development, which will be blocked (Rousseaux et al., Reference Rousseaux, Reynoird, Escoffier, Thevenon, Caron and Khochbin2008; Jenkins & Carrell, Reference Jenkins and Carrell2012). In fact, in order for the disulphide bridges to close (Fig. 2), it is necessary to have an initial stage of oxidation, thus the lipids in the membrane of the spermatozoon (I) will be peroxidized. Next, glutathione will be oxidized, converting to oxidized glutathione, which will regenerate the integrity of the spermatozoon membrane (II). Subsequently, the ox-glutathione will be reduced and regenerate reduced glutathione. The process is carried out by oxidation of protamine cysteines, forming disulphide bridges, appropriately ‘bundling up’ DNA (III). In other words, it is a cascade of three oxidation/reduction processes. Vitamin C is harmful at two levels, provided of course that its concentration increases in the serum and then in the seminal plasma. First, it prevents the primary oxidation level of the spermatozoon membrane (I), then later in phase III in which it is capable of opening disulphide bridges in the protamines, owing to its strong redox potential. Similar observations have been demonstrated by Donnelly et al. (Reference Donnelly, McClure and Lewis1999) and Giustarini et al. (Reference Giustarini, Dalle-Donne, Colombo, Milzani and Rossi2008): the capacity of vitamin C to reduce the disulfide bonds of proteins is a general concern. The concentration of vitamin C in blood is not a marker of oxidative stress (de la Villehuchet et al., Reference de la Villehuchet, Brack, Dreyfus, Oussar, Bonnefont-Rousselot, Chapman and Kontush2009; Brack et al., Reference Brack, Brack, Bonnefont-Rousselot, Dreyfus, Chapman and Kontush2012), either in endocrinological or neurovegetative diseases or even in cases of cancer (in 504 patients tested). For smokers, the first treatment with ascorbic acid was proposed by Fraga et al. (Reference Fraga, Motchnik, Shigenaga, Helbock, Jacob and Ames1991), using MDA as a control, however no difference in ascorbic acid concentration was found in the seminal plasma of smokers and non-smokers (Kand’ár et al., Reference Kand’ár, Drábková and Hampl2011).

Superoxide dismutase

SOD treatments lead to the same negative results as the association of selenium with the vitamins C, A and E (Ménézo et al., Reference Ménézo, Hazout, Panteix, Robert, Rollet, Cohen-Bacrie, Chapuis, Clément and Benkhalifa2007b). This type of treatment ‘swells’ the nucleus, undoubtedly in much the same way, in other words, via cystine bonds and opening of protamines and/or the prevention of the formation of cystine bridges that ‘lock up’ DNA.

Vitamins A and E

The association of these two vitamins seems to be effective in terms of the quality of spermatogenesis in vivo (Almbro et al., Reference Almbro, Dowling and Simmons2011). Vitamin A is generally given in the form of α-tocopherol. When it is used alone, it appears active in vivo; taking on an anti-inflammatory role, but it may have a negative effect on fertilization. It is effective in vitro when it is added during manipulation and freezing of spermatozoa. Vitamin E protects membranes. Once again, it is virtually impossible to find a correlation between beta-carotene content in plasma and endocrinological and other pathologies related to oxidative stress (Brack et al., Reference Brack, Brack, Bonnefont-Rousselot, Dreyfus, Chapman and Kontush2012). However, this event is not the case for vitamin E: endocrinological psychiatric pathologies and infectious syndromes may be linked to decreased serum concentration. Generally speaking, only vitamin E, when prescribed ‘haphazardly’, solely based on a spermiogram, without taking into account the quality of the DNA or the structure of the spermatozoon, seems to improve the number and/or the motility of spermatozoa.

Finally, when looking carefully at the scientific literature, treatment with beta-carotene, vitamin A, and vitamin E may increase mortality (Bjelakovic et al., Reference Bjelakovic, Nikolova, Gluud, Simonetti and Gluud2008) and dietary supplementation with vitamin E significantly increases the risk of prostate cancer among healthy men (Klein et al., Reference Klein, Thompson, Tangen, Crowley, Lucia, Goodman, Minasian, Ford, Parnes, Gaziano, Karp, Lieber, Walther, Klotz, Parsons, Chin, Darke, Lippman, Goodman, Meyskens and Baker2011).

Stimulation of synthesized endogenous glutathione: assisting the germinal cell line

Quercetin (Fig. 5), also known as quercetol, is a flavonoid of the flavonol type. Quercetin passes into the blood circulation and its metabolites have an extensive lifespan. It inhibits tumour necrosis factor (TNF) alpha and interleukin 8 involved in the inflammatory process. Glycosides that are found in certain fruits in their natural state are hydrolyzed by enzymes in the small intestine and are then absorbed. Metabolite glucuronide, sulphate and methyl, formed in the blood are slightly less active than the natural product, but have a prolonged lifespan (>10 h). It is known that quercetin has a positive impact on the germinal line, and more particularly on spermatozoa, observed in the late 1980s (Hammerstedt et al., Reference Hammerstedt, Volonté and Racker1988). It protects cells in spermatogenesis from pollutants (hydrocarbons, estrogenic hormones; Izawa et al., Reference Izawa, Kohara, Aizawa, Suganuma, Inakuma, Watanabe, Taya and Sagai2008; Li et al., Reference Li, Zhong, Jiang, Wang, Zuo and Sha2012). Moreover, when quercetin passes into the blood, it regulates and optimizes the response to oxidative stress in heavy smokers (Bøhn et al., Reference Bøhn, Myhrstad, Thoresen, Holden, Karlsen, Tunheim, Erlund, Svendsen, Seljeflot, Moskaug, Duttaroy, Laake, Arnesen, Tonstad, Collins, Drevon and Blomhoff2010). More mechanisms of action are being recognized. Foods that are rich in quercetin increase the synthesis of 15 ARN messengers that are involved in DNA repair mechanisms and four ARN messengers that are concerned with apoptosis (Bøhn et al., Reference Bøhn, Myhrstad, Thoresen, Holden, Karlsen, Tunheim, Erlund, Svendsen, Seljeflot, Moskaug, Duttaroy, Laake, Arnesen, Tonstad, Collins, Drevon and Blomhoff2010). It also regulates gamma-glutamylcysteine synthetase and glutathione synthetase expression, interacting directly on the promoters – the stage that limits synthesis of endogenous glutathione. It especially increases in the muscle (Moskaug et al., Reference Moskaug, Carlsen, Myhrstad and Blomhoff2005). Quercetin increases the endogenous natural defenses.

Betalains (Fig. 6)

We have seen that strong anti-oxidants have a rather negative and often random impact on spermatogenesis. However, it is also true that peroxidation of spermatozoa membrane lipids has a negative impact on mobility, especially when they are developing (Montjean et al., Reference Montjean, Ménézo, Benkhalifa, Cohen, Belloc, Cohen-Bacrie and de Mouzon2010). Betalains may be an appropriate answer to this problem through their protective effect on membrane lipids. Owing to their structure, they connect with membrane lipids and oxidize for them, then disengage. Because of their ability to carry out this task, they slow anarchistic cellular division and promote programmed cellular death of certain strains of cancerous cells in vitro (Sreekanth et al., Reference Sreekanth, Arunasree, Roy, Chandramohan Reddy, Reddy and Reddanna2007). In a sperm analysis, they significantly improve the mobility of the spermatozoa (Benkhalifa et al., Reference Benkhalifa, Cohen, Tosti, Cohen-Bacrie, Ballashova and Ménézo2008), which would support their protective effect on spermatozoon membrane lipids.

Figure 6 Chemical structure of betalains.

Betalains and quercetine are compounds of vegetable origin: one of the best sources being the prickly pear (Opuntia ficus-indica).

DNA fragmentation

Firstly, it is the current consensus that 30% is an upper tolerable level, whatever the technique applied. Beyond this limit, problems of fertility appear, insemination becomes more uncertain than in in vitro fertilization and ICSI (Evenson et al., Reference Evenson, Larson and Jost2002; Frydman et al., Reference Frydman, Prisant, Hesters, Frydman, Tachdjian, Cohen-Bacrie and Fanchin2008; Bungum et al., Reference Bungum, Bungum and Giwercman2011). Beyond 50%, the chances of conceiving are virtually inexistent. Our experience with Procrelia Men® (Nurilia France), a combination of Opuntia extract (quercetin and betalains, group B vitamins and zinc) gave the best results, with a treatment duration of a minimum of 4 months, thus covering a full spermatogenetic cycle. Initial results show a drop in fragmentation (Benkhalifa et al., Reference Benkhalifa, Cohen, Tosti, Cohen-Bacrie, Ballashova and Ménézo2008). When fragmentation is >50%, it may be useful to treat initially with a strong antioxidant mixture (Fertibiol®), which should not be administered for more than 1 month). This event is then followed by Condensyl®, treatment over the next 2 months, re-sustaining the capacity for a correct packaging of the nucleus. Our own experience with prolonged treatment with strong anti-oxidants (Astaxanthin, coQ10 in ubiquinol form), in the case of very high levels of fragmentation, showed a strong reduction in the production of spermatozoa (down to 5% of the initial value).

Absence of nuclear condensation

This parameter may be measured by SCSA or, more simply, by permeation with aniline blue. Here the current consensus is that above 25–30%, arrests embryonic development. The observations in ICSI of no fertilization even when it is clear that a spermatozoon has been correctly injected almost always correlate with this nuclear defect. For this type of problem, Condensyl®, which primarily contains group B vitamins and zinc is especially effective for this ‘non-fertile’ group of patients (Junca et al., Reference Junca, Gonzalez Marti, Tosti, Cohen, De la Fontaine, Benkhalifa and Ménézo2012), and notably in couples, in who repeated ICSI and intracytoplasmic morphologically selected sperm injection (IMSI) were unsuccessful. In a short experiment with 27 patients, a 32% increase in condensation was observed (p < 0.0002) in association with eight pregnancies, of which five were spontaneous and three were following an ART attempt (Ménézo et al., in press). Malone dialdehyde formation, a ‘marker of oxidative stress’, is correlated strongly with sperm nucleus decondensation/immaturity (Montjean et al., Reference Montjean, Ménézo, Benkhalifa, Cohen, Belloc, Cohen-Bacrie and de Mouzon2010) and could be treated with this approach.

Recycling homocysteine i.e. the one-carbon cycle

Many studies have shown that ovarian stimulation increases homocysteine concentration in follicular liquid (Ebisch et al., Reference Ebisch, Peters, Thomas, Wetzels, Peer and Steegers-Theunissen2006, Reference Ebisch, Thomas, Peters, Braat and Steegers-Theunissen2007; Berker et al., Reference Berker, Kaya, Aytac and Satiroglu2009). Hcy passes into the oocyte and impedes methylation while competing with methionine (Ménézo et al., Reference Ménézo, Khatchadourian, Gharib, Hamidi, Greenland and Sarda1989). As a result, there is a major risk of creating a breeding ground for imprint type disorders, among which, epigenetic anomalies (Ménézo et al., Reference Ménézo, Mares, Cohen, Brack, Viville and Elder2011b). Therefore compounds such as Zn and the entire group of B vitamins that promote homocysteine recycling, are most welcome (either in vitro or in vivo during early embryo culture). In the animal kingdom, it is important to remember that optimization of processes of methylation supports genetic stability (Cooney et al., Reference Cooney, Dave and Wolff2002) by promoting prolificacy and the quality of pups obtained. Up to J3 post conception, the human oocyte and the very young embryo are not fully equipped to recycle homocysteine, the cystathionine beta synthase (CBS) pathway is absent (Benkhalifa et al., Reference Benkhalifa, Montjean, Cohen-Bacrie and Ménézo2010) and the betaine-homocysteine methyl transferase (BHMT) pathway is expressed weekly. This situation also limits the endogenous synthesis of glutathione, which implies that, in addition to folic acid, vitamins B2, B6 and B12 can be limiting. Medically assisted procreation clearly demonstrates the problem of methylation (Katari et al., Reference Katari, Turan, Bibikova, Erinle, Chalian, Foster, Gaughan, Coutifaris and Sapienza2009; Ménézo et al., Reference Ménézo, Elder, Benkhalifa and Dale2010b). Finally, with regard to the quality of DNA, we quote Lopes’ and Zenzes’ findings (Lopes et al., Reference Lopes, Jurisicova and Casper1998; Zenzes et al., Reference Zenzes, Puy and Bielecki1998; Zenzes, Reference Zenzes2000) that damage to DNA may affect the oocyte which is not really protected in ovary, during quiescence and in the growing follicle. In smokers, tar creates the formation of adducts with DNA, detected in ovarian cells and the preimplantation embryo. Therefore, we recommend the same type of preventive treatment (e.g. Procrelia® women) that contains polyphenols, quercetin, betalains, group B vitamins and zinc, at the outset of ART.