Wolf-Bernhard Schill

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.

ω-3 Fatty acid–based lipid infusion in patients with chronic plaque psoriasis: Results of a double-blind, randomized, placebo-controlled, multicenter trial

Abstract Background: Profound changes in the metabolism of eicosanoids with increased concentrations of free arachidonic acid (AA) and its proinflammatory metabolites have been observed in psoriatic lesions. Free eicosapentaenoic acid (EPA) may compete with liberated AA and result in an antiinflammatory effect. Objective: Our purpose was to assess the efficacy and safety of intravenously administered fish-oil–derived lipid emulsion on chronic plaque-type psoriasis. Methods: A double-blind, randomized, parallel group study was performed in eight European centers. Eighty-three patients hospitalized for chronic plaque-type psoriasis with a severity score of at least 15 according to the Psoriasis Area and Severity Index (PASI) participated in a 14-day trial. They were randomly allocated to receive daily infusions with either a ω-3 fatty acid–based lipid emulsion (Omegavenous; 200 ml/day with 4.2 gm of both EPA and docosahexaenoic acid (DHA); 43 patients) or a conventional ω-6-lipid emulsion (Lipovenous; EPA+DHA Results: The total PASI score decreased by 11.2 ± 9.8 in the ω-3 group and by 7.5 ± 8.8 in the ω-6 group ( p = 0.048). In addition, the ω-3 group was superior to the ω-6 group with respect to change in severity of psoriasis per body area, change in overall erythema, overall scaling and overall infiltration, as well as change in overall assessment by the investigator and self-assessment by the patient. Response (defined as decrease in total PASI of at least 50% between admission and last value) was seen in 16 of 43 patients (37%) receiving the ω-3 emulsion and 9 of 40 patients (23%) receiving ω-6 fatty acid–based lipid emulsion. No serious side effects were observed. Within the first few days of ω-3 lipid administration, but not in the ω-6 supplemented patients, a manifold increase in plasma-free EPA concentration, neutrophil leukotriene B 5 and platelet thromboxane B 3 generation occurred. Conclusion: Intravenous ω-3-fatty acid administration is effective in the treatment of chronic plaque-type psoriasis. This effect may be related to changes in inflammatory eicosanoid generation. (J Am Acad Dermatol 1998;38:539-47.)

The male reproductive system and its susceptibility to endocrine disrupting chemicals

Summary.  In the past years, there has been increased interest in assessing the relationship between impaired male fertility and environmental factors. Human male fertility is a complex process and therefore a great variety of sites may be affected by exogenous noxae. Lifestyle factors as well as various environmental and occupational agents may impair male fertility. Many studies have been published reporting on reproductive dysfunctions in male animals and humans. Especially environmental pollutants with endocrine activity are discussed as a possible cause of this detrimental development. Evidence from animal experiments show that substances with oestrogenic and antiandrogenic properties may cause hypospadia, cryptorchidism, reduction of sperm density and an increase of testicular tumours. Many adverse effects on animal male fertility have been documented for phthalates and some chlorinated hydrocarbons such as polychlorinated biphenyls and polychlorinated dibenzo‐p‐dioxins. For other chemicals such as bisphenol A and nonylphenols animal data are conflicting. Environmental pollutants may mediate their effects by receptor binding, modulation of hormone‐regulated mechanisms or direct toxic effects. Data on environmental chemicals and human male fertility are scarce, and risk assessment is mostly based on the results of animal studies. However, there are indications that exposure to endocrine active chemicals during early development may alter hormone responsiveness in adulthood. Furthermore, some of the chemicals are found in fluids that are associated with human reproduction, such as follicular fluid, seminal fluid and cervical mucus. Recent studies suggest a correlation between pesticide exposure and standard semen parameters as well as in vitro fertilization rates.

Sertoli cells as a target for reproductive hazards

Male fertility can be impaired by various toxicants. Some of them are known to target mainly Sertoli cells, which play an essential role in spermatogenesis. In this study, the in vitro response of immature rat Sertoli cells to various environmental pollutants, including pesticides, oestrogenic compounds and heavy metals, has been investigated. Mitochondrial dehydrogenase activity has been used to measure Sertoli cell viability, while production of lactate and secretion of inhibin B have been used as general and specific cell markers. Sertoli cell viability was not affected after 24‐h exposure to lindane, DDT, ethinyloestradiol or bisphenol A in the concentration range analysed (up to 100, 25 or 50 μm, respectively). In contrast, mercury(II) (EC50=31 μm) and cisplatin (15% decrease in viability at 100 μm) induced some cytotoxic effect. With the exception of the pesticide DDT, all chemicals investigated induced a significant dose‐dependent increase in lactate production after 24‐h exposure to Sertoli cells. Owing to the cytotoxic effect of mercury(II), lactate levels dropped again at concentrations above 20 μm. The pesticide lindane (but not DDT) and both oestrogens significantly increased the production of the Sertoli cell specific hormone inhibin B without affecting cell viability. In contrast, the heavy metals mercury(II) and platinum(II) markedly decreased inhibin B levels. This sharp decrease was already significant at metal concentrations that reduced Sertoli cell viability only moderately (10–15%). In conclusion, the secretion of lactate and inhibin B by immature rat Sertoli cells seems to be a useful and sensitive marker with which to explore potential Sertoli cell toxicants.

Influence of urogenital infections on sperm functions

Summary. Many studies have examined the impact of genital tract infections on male fertility; however, the effect of bacteriospermia on sperm quality is still controversial. Bacterial infections are more frequently found in semen samples from asymptomatic infertile patients than in those from fertile men. Bacteriospermia is also a common problem of male partners from couples undergoing IVF. Therefore, the effects of microorganisms on human sperm acrosome reaction of oocytes have been studied in vitro and in vivo.

Ageing and sperm function.

To evaluate the fertilizing capacity of spermatozoa from elderly men, ejaculates from 29 older fathers (mean age 50.3 years) were compared with those from 35 younger fathers (mean age 32.2 years). In addition to conventional semen parameters, sperm functions were studied that have been reported to be positively correlated with the fertilization rate: progressive motility, acrosin activity, inducible acrosome reaction, and chromosome condensation. Sperm concentration and follicle stimulating hormone concentration differed significantly in both groups. With regard to sperm functions there were no differences between older men and younger men, except for decreased sperm motility in the older group which, however, reached nearly normal values according to World Health Organization criteria. Decreased fertility of older couples is obviously more dependent on the age of the female partner. The significance of genetic risks remains to be clarified, especially when methods of assisted reproduction are applied.

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.

Lichen amyloidosus: A consequence of scratching?

BACKGROUND Lichen amyloidosus (LA) is generally said to be a pruritic type of amyloidosis of unknown cause. Histopathologically, it is characterized by epidermal changes of lichen simplex chronicus and by deposits of amyloid in the papillary dermis that are derived from keratin peptides of necrotic keratinocytes. Chronic scratching is responsible for the development of lichen simplex chronicus and may lead to necrosis of individual keratinocytes. OBJECTIVE Our purpose was to evaluate whether chronic scratching may also be responsible for the formation of amyloid in LA. METHODS We studied patients with LA in regard to histopathologic findings, onset of pruritus, associated diseases, and response to treatment. RESULTS In most cases, pruritus had preceded the skin lesions. Eight of nine patients suffered from diseases other than LA that may be associated with pruritus. Histopathologically, amyloid was confined to areas that also showed signs of lichen simplex chronicus. Systemic treatment with sedating antihistamines and intense local treatment with corticosteroids were found to be effective. CONCLUSION LA is considered to be a variant of lichen simplex chronicus in which scratching leads to necrosis of keratinocytes and eventually to the formation of amyloid in the papillary dermis. Because chronic scratching seems to be the cause and not the result of the deposits of amyloid, treatment should be directed at the amelioration of pruritus.

Spiradenomas in Brooke-Spiegler syndrome

Brooke—Spiegler syndrome is an autosomal duminantly inherited disease characterized by the development of multiple trichoepitheliomas and cylindromas. Among other neoplasms that may also occur in Brooke—Spiegler syndrome are basal cell carcinomas and spiradenomas. Spiradenomas and cylindromas have so many features in common that they have been regarded as variants of the same neoplasm. This assumption was supported by the occurrence of both types of lesions in Brooke—Spiegler syndrome. We report a case of Brooke—Spiegler syndrome in which spiradenomas were found in the immediate vicinity of trichoepitheliomas and in continuity with follicles. Because of the embryonic relationship between follicles and apocrine glands, these features indicate that spiradenomas are apocrine neoplasms. We conclude that Brooke—Spiegler syndrome is an inherited disease that affects the folliculosebaceous apocrine unit.