Ralf Henkel

Sperm preparation for ART

The onset of clinical assisted reproduction, a quarter of a century ago, required the isolation of motile spermatozoa. As the indication of assisted reproduction shifted from mere gynaecological indications to andrological indications during the years, this urged andrological research to understand the physiology of male germ cell better and develop more sophisticated techniques to separate functional spermatozoa from those that are immotile, have poor morphology or are not capable to fertilize oocytes. Initially, starting from simple washing of spermatozoa, separation techniques, based on different principles like migration, filtration or density gradient centrifugation evolved. The most simple and cheapest is the conventional swim-up procedure. A more sophisticated and most gentle migration method is migration-sedimentation. However, its yield is relatively small and the technique is therefore normally only limited to ejaculates with a high number of motile spermatozoa. Recently, however, the method was also successfully used to isolate spermatozoa for intracytoplasmic sperm injection (ICSI). Sperm separation methods that yield a higher number of motile spermatozoa are glass wool filtration or density gradient centrifugation with different media. Since Percoll® as a density medium was removed from the market in 1996 for clinical use in the human because of its risk of contamination with endotoxins, other media like IxaPrep®, Nycodenz, SilSelect®, PureSperm® or Isolate® were developed in order to replace Percoll®. Today, an array of different methods is available and the selection depends on the quality of the ejaculates, which also includes production of reactive oxygen species (ROS) by spermatozoa and leukocytes. Ejaculates with ROS production should not be separated by means of conventional swim-up, as this can severely damage the spermatozoa. In order to protect the male germ cells from the influence of ROS and to stimulate their motility to increase the yield, a number of substances can be added to the ejaculate or the separation medium. Caffeine, pentoxifylline and 2-deoxyadenosine are substances that were used to stimulate motility. Recent approaches to stimulate spermatozoa include bicarbonate, metal chelators or platelet-activating factor (PAF). While the use of PAF already resulted in pregnancies in intrauterine insemination, the suitability of the other substances for the clinical use still needs to be tested. Finally, the isolation of functional spermatozoa from highly viscous ejaculates is a special challenge and can be performed enzymatically to liquefy the ejaculate. The older method, by which the ejaculate is forcefully aspirated through a narrow-gauge needle, should be abandoned as it can severely damage spermatozoa, thus resulting in immotile sperm.

DNA fragmentation of spermatozoa and assisted reproduction technology

Despite the ever-increasing knowledge of the fertilization process, there is still a need for better understanding of the causes of sperm DNA fragmentation and its impact on fertilization and pregnancy. For this reason, human sperm DNA fragmentation was investigated by means of the terminal deoxynucleotidyl transferase-mediated dUDP nick-end labelling (TUNEL) assay and the production of reactive oxygen species (ROS) in the ejaculate and in the spermatozoa themselves. These data were correlated with fertilization and pregnancy data from IVF and intracytoplasmic sperm injection (ICSI) patients. Sperm DNA fragmentation did not correlate with fertilization rate, but there was a significantly reduced pregnancy rate in IVF patients inseminated with TUNEL-positive spermatozoa. ICSI patients exhibited the same tendency. This implies that spermatozoa with damaged DNA are able to fertilize an oocyte, but at the time the paternal genome is switched on, further development stops. The determination of ROS in the ejaculate and the percentage of ROS-producing spermatozoa revealed markedly stronger correlations between sperm functions (i.e. motility) and the percentage of ROS-producing spermatozoa. The influence of seminal leukocytes, known to produce large amounts of oxidants, on sperm DNA fragmentation should not be neglected.

Relevance of zinc in human sperm flagella and its relation to motility

OBJECTIVE To measure the zinc content of human sperm flagella and to analyze its relation to sperm motility. DESIGN Prospective study. SETTING Center of Dermatology and Andrology. PATIENT(S) Semen samples collected from 90 andrology patients and healthy donors after 3-5 days of sexual abstinence. INTERVENTION(S) None. MAIN OUTCOME MEASURE(S) Sperm morphology after Shorr staining, sperm motility, and patient age were recorded. In addition, zinc concentrations in the seminal plasma, sperm heads, and flagella were determined with the use of atomic absorption spectrometry. RESULT(S) The mean zinc concentration was 144.3 mg/L in the seminal plasma and 146.9 mg/L in the whole ejaculate and was significantly correlated with parameters of motility. The sperm heads contained only 6.7% of the zinc that was present in the whole spermatozoon. The zinc concentration in the flagella was negatively correlated with sperm motility and velocity. In addition, it was positively correlated with the percentage of abnormally blue-stained flagella and the age of the patients. CONCLUSION(S) Our results clearly demonstrate the importance of zinc elimination during epididymal sperm maturation for functional competence of the outer dense fibers and, therefore, generation of motility.

Update on the impact of Chlamydia trachomatis infection on male fertility.

With approximately 90 million cases annually, infection with Chlamydia trachomatis is the most prevalent sexually transmitted bacterial disease in the world. Considering that these infections are often asymptomatic and cause major complications like acute pelvic inflammatory disease, ectopic pregnancy, infertility or infant pneumonia, the estimated costs for diagnosis and treatment in the USA amounts to 2.2 million US dollars for each 500 cases. Therefore, there is a high need for correct, quick and cost‐effective diagnosis and treatment of this urogenital tract infection. New innovative therapies provide good results with regard to efficacy and patients’ compliance. The success rates of treatments are at least 95%. However, the occurrence of antibiotic resistance should not be ignored and new treatment schemes must be developed. The state‐of‐the‐art of diagnosis and treatment of chlamydial infections as well as the pathophysiology is discussed in this review. In conclusion, infections with C. trachomatis is an important public health problem, especially in third world and developing countries, and more socio‐economic studies linking secondary prevention of chlamydial infections, infertility and adverse pregnancy outcome are needed to understand more of its aetiology. In addition, diagnosis and treatment should be improved. Data in men revealed that past infections but not present infections are more related to male infertility. There is still controversial results. In future studies, function of the seminal vesicles and evaluation of the antioxidant capacity should be taken into account when role of C. trachomatis infection on male fertility is assessed.

Scavenging effect of N‐acetyl‐L‐cysteine against reactive oxygen species in human semen: a possible therapeutic modality for male factor infertility?

Summary. A new approach to reduce the level of reactive oxygen species (ROS) in human semen by using N‐acetyl‐L‐cysteine (NAG) was evaluated. Semen samples were incubated with or without NAC (1.0 nig ml−1) at room temperature. The chemiluminescent signal of the oxidation of luminol was detected by means of an MTP reader after 0, 20, 40, 60 and 120 min, respectively, using 200 μM luminol. In addition, the dose‐dependent action of NAC (0.1, 1.0 and 5.0 mg ml−1) and the influence of NAC on functional sperm parameters (motility and acrosome reaction) were studied.

The impact of oxidants on sperm function

Fertilization and pregnancy are dependent on a series of functional sperm parameters, which are reportedly affected by reactive oxygen species (ROS) like hydrogen peroxide (H2O2), superoxide anion (O 2 ) and/or hydroxyl radical (OH) (Aitken et al., 1989). These highly reactive substances, which exhibit half-life times in the nanosecond (OH) to the milli-second range (O 2 ), are very strong oxidants and are physiologically produced in any living cell during respiration. Because of the extraordinarily high content of polyunsaturated fatty acids in the plasma membrane, which is an essential requirement for the male germ cell to maintain sperm functions, and due to the very low content of protective systems, spermatozoa are highly susceptible to oxidative stress (Aitken et al., 1989). This has repeatedly been shown to be a cause of impaired sperm function and thus male infertility (Plante et al., 1994). However, it was shown that ROS also play a key role in the control of sperm function, for instance by redox regulation of tyrosine phosphorylation (Ecroyd et al., 2003), which is intimately involved in signal transduction of capacitation and acrosome reaction. Thus, the levels between these physiologically essential ROS and an excessive production of ROS in spermatozoa must be finely balanced. Moreover, an exposure of spermatozoa to high amounts of ROS during the passage through the male genital tract system or in the semen must be avoided. In the male reproductive system, ROS can derive from leucocytes or from the sperm cells themselves. In the ejaculate, spermatozoa have been repeatedly shown to be only a minor source of ROS production while infiltrating or contaminating leucocytes (peroxidase-positive cells) are the predominant source of these oxidants. Leucocytes are present in almost any ejaculate and produce at least 1000times more ROS than spermatozoa (de Lamirande & Gagnon, 1995) within the scope of their normal physiological function, immunosurveillance and thus immunological defence with elimination of pathogenic germs. However, in the case of inflammations, the production of ROS within the genital tract system is very much elevated and can deprive the anti-oxidative protection system which is provided by the seminal fluid and normally contains high amounts of antioxidants like spermin, thiols, uric acid or vitamin C, thus causing oxidative stress to the spermatozoa. However, oxidative stress to spermatozoa can also occur if the seminal concentration of antioxidants is pathologically decreased. In infertile men, the anti-oxidative capacity has been shown to be significantly lower and the concentration of ROS higher than in fertile subjects. According to the World Health Organization, male genital tract inflammation is defined as significant bacteriospermia, detection of Neisseria gonorrhoeae, Chlamydia trachomatis or Ureaplasma urealyticum or significant leucocytospermia (>1 · 10 leucocytes/ml). Direct effects of bacteria on human spermatozoa comprise adhesion, agglutination and morphological alterations. In addition, direct and indirect negative effects of bacteria on sperm motility and acrosome reaction have been observed. The effects of leucocytes, however, appear to be mediated by cytokines and ROS, the latter of which have been shown to affect acrosome reaction, acrosin activity and sperm DNA fragmentation. In this context, the question arises whether the influence of ROS produced by leucocytes or the sperm cells themselves affect sperm functions in nonleucocytospermic patients. Moreover, it is of clinical interest for assisted reproduction programmes to know whether or not these two sources of ROS act differently on male germ cells. Therefore, we examined ROS production in the ejaculate and by the spermatozoa themselves as well as sperm DNA fragmentation in 63 non-leucocytospermic patients. Additionally, motility, count, motility percentage before and after swim-up and normal sperm morphology as well as the number of peroxidasepositive cells were recorded. Leucocytes were highly significantly correlated with the ROS production in the ejaculate (r 1⁄4 0.576; P < 0.0001) but markedly less with intrinsic ROS production (r 1⁄4 0.296; P 1⁄4 0.0218). Sperm count, morphology and motility in the ejaculate were markedly more affected by extrinsic than by intrinsic ROS. Furthermore, DNA fragmentation was positively correlated with intrinsic ROS production. This correlation was weaker for ROS produced by leucocytes. The correlation between motility

Sperm separation in patients with urogenital infections

Summary. A total of 196 patients attending the Center of Dermatology and Andrology, Giessen, Germany, were examined for fertility problems. Polymorphonuclear elastase, reactive oxygen species (ROS) and the number of round and peroxidase‐positive cells were investigated in addition to routine semen analysis. The ejaculates were also analysed before and after sperm separation by means of swim‐up or glass wool filtration. In 20 cases of leukocytospermia, sperm concentration, motility, viability, production of reactive oxygen species, and the number of peroxidase‐positive cells were evaluated before and after glass wool filtration. The results show that ROS production by viable spermatozoa is highly correlated with the concentration of PMN elastase and the number of both peroxidase‐positive and round cells. Multiple regression analysis with motility as dependent parameter showed the number of round cells (n = 91; r = ‐0.332; P = 0.0030) to be the most important parameter affecting motility, while ROS mainly affects the viability of spermatozoa (n = 69; r = 0.250; P = 0.0107). In the case of leukocytospermia, glass wool filtration significantly reduced the number of peroxidase‐positive cells and ROS production (P = 0.0098 and P = 0.0005, respectively). Receiver operating characteristic (ROC) curve analysis for ROS production in the ejaculate using a concentration of 1.000 ng ml−1 PMN elastase as decisive parameter resulted in a cut‐off value of 49,489.9 counts 10−7 viable spermatozoa. The statistical parameters were: Sensitivity: 63.2%, specificity: 100%, positive predictive value: 100%, negative predictive value: 36.1%.

TUNEL assay and SCSA determine different aspects of sperm DNA damage.

For the determination of sperm DNA damage, different assays are used. However, no further distinction is made and the literature generally speaks about DNA damage. Thus, this study aimed at comparing the sperm chromatin structure assay (SCSA) and the TUNEL assay. In 79 patients, sperm DNA damage was determined flow cytometrically using the SCSA and the TUNEL assay. Moreover, normal sperm morphology was evaluated according to strict criteria. A statistical comparison of the two methods was performed using standard correlations, Bland and Altman plots, Passing–Bablok regressions and concordance correlation. Results show a significant difference between P‐ and G‐pattern morphology only for the mean channel fluorescence of the SCSA. Spearman’s rank correlations between the different parameters of both assays, SCSA and TUNEL, revealed significant associations between the parameters of the assays. However, when applying Bland and Altman plots, Passing–Bablok regression and concordance correlation results showed that these methods are not comparable. These different techniques determine different aspects of sperm DNA damage, i.e. ‘real’ DNA damage for the TUNEL assay and ‘potential’ DNA damage in terms of susceptibility to DNA denaturation for the SCSA. Thus, one should clearly distinguish between the different assays, not only practically and methodologically but also linguistically.

Urogenital inflammation: changes of leucocytes and ROS

Summary. The presence of excess leucocytes in the semen has been associated with male infertility. According to the WHO, concentrations of more than 106 leucocytes ml−1 are considered as leucocytospermia, indicating genital tract infections. Up to now, no consensus has been achieved on how leucocytes should be quantified in semen. Using the peroxidase staining and monoclonal antibodies to CD15, CD45 and CD68, we found significant differences between the detection methods. Only 47.4% of the semen samples that were assessed as leucocytospermic by CD45 were identified as such by peroxidase staining. The concentration of peroxidase‐positive cells was significantly correlated with polymorphonuclear granulocyte (PMN) elastase (P < 0.0001). However, a negative correlation of peroxidase‐positive cells with the sperm concentration was only found in oligozoospermic patients (P < 0.0001). Moreover, the slightly positive correlation with normal sperm morphology seems to be applicable only in cases of oligozoospermia. Significant negative correlation of the number of peroxidase‐positive cells were found for both maximal inducible acrosome reaction (P = 0.0219) and the inducibility of acrosome reaction (P = 0.0370), indicating a rather deleterious effect of leucocytes on this important sperm function. Concerning the result in the in vitro fertilization programme, none of the examined parameters (PMN elastase, concentration of round cells and peroxidase‐positive cells) showed a correlation with either fertilization or pregnancy. This result seems to be reasonable as severely damaged spermatozoa and leucocytes are eliminated from the ejaculate by different sperm separation methods. Interestingly, a significant negative correlation of the TUNEL assay as a measure of sperm DNA fragmentation was found only with pregnancy (P = 0.006) but not with fertilization. As DNA fragmentation can also be caused by ROS that are generated by leucocytes, this causality should not be neglected.

Standardised water‐soluble extract of Eurycoma longifolia, Tongkat ali, as testosterone booster for managing men with late‐onset hypogonadism?

In most countries, millions of people are relying on herbal medicines as remedy for numerous ailments. In South‐East Asia, Eurycoma longifolia Jack, also known as ‘Malaysian ginseng’ or Tongkat ali, is used to combat stress and disease and to improve physical strength. Moreover, the compounds of the roots of this plant are reported to have aphrodisiac and testosterone enhancing effects in the rat. Considering that human studies are not available, 76 of 320 patients suffering from late‐onset hypogonadism (LOH) were given 200 mg of a standardised water‐soluble extract of Tongkat ali for 1 month. The Ageing Males’ Symptoms (AMS) according to the standardised rating scale and the serum testosterone concentration were taken. Results show that treatment of LOH patients with this Tongkat ali extract significantly (P < 0.0001) improved the AMS score as well as the serum testosterone concentration. While before treatment only 10.5% of the patients did not show any complaint according to the AMS scale and 35.5% had normal testosterone levels, after the completed treatment 71.7% and 90.8% of the patients showed normal values, respectively. Thus, Tongkat ali extract appears to be useful as a supplement in overcoming the symptoms of LOH and for the management of hypogonadism.