Hans-Juergen Glander

Obesity and age affect male fertility potential

A case-cohort study of 2,157 patients, aged 17-67 years was performed to assess the impact of body mass index and age on male fertility. Using a multiple regression analysis across the total cohort, only age correlated significantly with all spermiogram parameters and serum hormones, but in patients aged 20-30 years (n = 617) the total sperm count was significantly negatively correlated with body mass index.

Evaluation of sperm recovery following annexin V magnetic-activated cell sorting separation

Magnetic-activated cell sorting (MACS) using paramagnetic annexin V-conjugated microbeads eliminates spermatozoa with externalized phosphatidylserine, which is considered one of the features of apoptosis. The objective of this study was to evaluate sperm recovery following the use of MACS as a sperm preparation technique. Mature spermatozoa were separated and divided into two fractions: the first was prepared by density gradient centrifugation (DGC) and MACS, while the second was prepared by DGC only. Following MACS, the percentage of cells collected in the annexin-negative fraction was significantly higher than the annexin-positive fraction and the sperm recovery rate was 73.8 +/- 12.1%. In conclusion, the integration of MACS with DGC can be considered as an effective sperm preparation technique that does not lead to significant cell loss. Separating a distinctive population of non-apoptotic spermatozoa with intact membranes may optimize the outcome of assisted reproduction.

Capacitation and acrosome reaction in nonapoptotic human spermatozoa.

Abstract:  Capacitation and acrosome reaction (AR) of human spermatozoa are prerequisites for fertilization. Annexin‐V‐MACS is able to separate apoptotic from nonapoptotic sperm on the basis of their externalization of phosphatidylserine (EPS). The nonapoptotic (EPS−) fraction is characterized by the lowest amounts of membrane alterations, caspase activation, disrupted mitochondrial potential, and DNA fragmentation. The aim of our study was to investigate the separation effect of Annexin‐V‐MACS on capacitation and AR in nonapoptotic sperm. Semen specimens from 10 healthy donors were separated into 2 samples each, one was left untreated (control) and the second was subjected to Annexin‐V‐MACS. Two aliquots of both, the control as well as the EPS− fraction after Annexin‐V‐MACS, were incubated in HTF at 37°C, 5% CO2 for 3 h either with 3% BSA (capacitation) or without additives. Capacitation was monitored by tyrosine phosphorylation (TyrP) using Western blot technique. AR was determined by labeling with CD46‐FITC before and after stimulation with calcium‐ionophore A23187, followed by flow cytometric evaluation of the percentage of CD46+ sperm. Densitometric analyses of the 105‐kDa and 80‐kDa bands of the TyrP Western blots demonstrated highest TyrP in the capacitated EPS− aliquots. There was no difference in spontaneous AR in all groups. AR was best inducible in EPS‐negative sperm after capacitation. Nonapoptotic human spermatozoa with intact plasma membranes are characterized by superior ability to capacitate and consequently by maximum potential to perform AR after stimulation. Selection of EPS‐negative sperm may be of advantage for assisted reproduction in order to prepare the sperm subpopulation with the highest fertilizing potential.

Mitochondria of human spermatozoa are preferentially susceptible to apoptosis.

Abstract: Although elements of type I and II apoptosis have been demonstrated in human spermatozoa, their functionality has not been evaluated. The first dynamic studies revealed no type I apoptosis signal transduction via CD95. The aim of our study was to clarify whether type II apoptosis can be induced in human spermatozoa. Betulinic acid, a cytotoxic agent and highly specific inductor of type II apoptosis, acts by a direct effect on mitochondria. Motility, mitochondrial transmembrane potential, and active caspase‐9 and ‐3 were examined in human ejaculated spermatozoa of 33 semen samples from healthy volunteers after incubation with 60 μg/mL betulinic acid for 10 and 60 min, respectively. Untreated aliquots of each sample served as negative controls. Treatment with betulinic acid resulted in the induction of type II apoptosis measured by disruption of mitochondrial transmembrane potential and activation of caspase‐9 and ‐3. The loss of mitochondrial energy supply resulted in a significant decrease of spermatozoal motility and velocity. In spermatozoa, mitochondria are tightly packed and located exclusively in the midpiece region. This might contribute to their susceptibility against the induction of type II apoptosis and should be considered for therapeutic interventions and might have a future in the development of advanced birth control methods.

Increased sperm chromatin decondensation in selected nonapoptotic spermatozoa of patients with male infertility

OBJECTIVE To evaluate the sperm chromatin decondensation (SCD) rates of the annexin-negative (nonapoptotic) sperm fraction of patients with infertility using hamster intracytoplasmic sperm injection (H-ICSI). In healthy donors, the depletion of apoptotic sperm using annexin V-based magnetic-activated cell separation (MACS) enhances hamster oocyte sperm penetration but does not increase SCD rates following H-ICSI. DESIGN A prospective-controlled study. SETTING Male infertility clinic, European Academy of Andrology Center Leipzig. PATIENT(S) Twenty-one male infertility patients with subnormal spermiogram. INTERVENTION(S) Spermatozoa were separated by Annexin V-MACS. MAIN OUTCOME MEASURE(S) Apoptosis signaling (disruption of transmembrane mitochondrial potential, transmembrane mitochondrial potential [TMP], and activation of caspases-3 [CP3]) and SCD rates of human spermatozoa after hamster intracytoplasmic sperm injection. RESULT(S) Infertility patients showed high levels of sperm with active CP3 and disrupted TMP, which correlated negatively with SCD rates. Annexin V-MACS resulted in a significant enrichment of spermatozoa with inactive CP3 and intact TMP in the annexin-negative fraction. Similarly, annexin-negative sperm had the highest SCD rates following H-ICSI compared with controls and annexin-positive sperm. CONCLUSION(S) These results suggest that nonapoptotic spermatozoa prepared by annexin V-MACS display higher early fertilization potential following ICSI. The technique should be evaluated in a clinical setting for its impact on ICSI outcomes in patients diagnosed with infertility.

Magnetic-activated cell sorting before cryopreservation preserves mitochondrial integrity in human spermatozoa

Superparamagnetic annexin-V conjugated microbeads are able to eliminate spermatozoa with externalized phosphatidylserine, a membrane feature of apoptotic cells as well as spermatozoa with deteriorated plasma membrane. Our objective was to evaluate the effects of annexin-V Magnetic-Activated Cell Sorting (MACS) in cryopreservation–thawing protocols and on integrity of sperm mitochondrial transmembrane potential and mitochondrial integrity survival rate (MSR). Mature spermatozoa of 10 healthy donors were prepared by density gradient centrifugation and divided into 2 aliquots afterwards. The first one was subjected to annexin-V MACS followed by cryopreservation and thawing, while the second was cryopreserved–thawed without MACS to serve as control. Annexin-negative sperm separated by MACS showed significantly higher levels of intact mitochondria following cryopreservation–thawing (45.4±8.6%) compared to sperm that were not separated (15.8±4.6%, p<0.01). Separating a distinctive population of non-apoptotic spermatozoa with intact membranes may optimize cryopreservation–thawing outcome. MACS using annexin-V microbeads enhances the percentage of spermatozoa with intact transmembrane mitochondrial potential and mitochondrial integrity survival rates following cryopreservation.

Impact of caspase activation in human spermatozoa

Caspases are central components in the apoptosis signaling cascade. The family of cysteine proteases transduces and enhances the apoptosis signal, and activation of effector caspases results in controlled cellular degradation. Although initially the presence of caspases in spermatozoa was controversially discussed in recent years, many studies demonstrated their activation in male germ cells. Activated apoptosis signaling results in decreased fertilizing capacity of the sperm. This review presents the current knowledge on the role of caspases in human sperm. Techniques of caspase monitoring are highlighted. With regard to the high impact of caspases on the sperm fertilizing potential, physiological and pathological settings of caspase activation and inactivation are discussed. Finally, the effects of depletion of caspase‐positive sperm are shown with various standard and molecular sperm preparation methods. Microsc. Res. Tech., 2009.

Sperm apoptosis signalling in diabetic men

Male patients with diabetes type I and type II present more frequently with subfertility. On a subcellular level, increased apoptosis signalling and the rate of DNA fragmentation have an impact on sperm fertilizing capacity. The aim of this study was to evaluate apoptosis signalling and the role of DNA fragmentation in sperm of patients with diabetes type I and type II to gain further insight into the pathophysiology of diabetes-related subfertility in men. Semen specimens collected from 18 healthy fertile donors and 27 donors with diabetes type I (n=13) or type II (n=14) were prepared via density gradient centrifugation. High- and low-density sperm subpopulations were assessed for apoptosis markers (disrupted transmembrane mitochondrial potential, activated caspase 3) and reactive oxygen species, as well as DNA fragmentation, by flow cytometry. The results show that ejaculates of diabetic men contain significantly (P<0.05) higher concentrations of spermatozoa with disrupted transmembrane mitochondrial potential, activated caspase 3, reactive oxygen species and fragmented DNA when compared with healthy fertile donors. The effect is more pronounced in men with diabetes type II. All measured parameters were inversely correlated with the sperm fertilizing potential, indicating a possible mechanism of subfertility in diabetic men. Diabetes mellitus can affect male fertility. Earlier studies have proved reduced sperm motility and lower DNA integrity in germ cells of diabetic patients. It was postulated that higher levels of oxidative stress may contribute to this findings. However, until now the pathophysiology of diabetes-related male subfertility is not fully understood. Our study showed for the first time that apoptosis signalling, measured by disrupted transmembrane mitochondrial potential and activated caspase 3, is significantly increased in sperm from males with diabetes type-I and type-II. Particularly, a disrupted transmembrane mitochondrial potential contributes to the reduced sperm motility. Together with the increased presence of intracellular reactive oxygen species and higher levels of sperm DNA fragmentation, several subcellular factors are now available to explain subfertility in diabetic males.

Age-dependent inhibin B concentration in relation to FSH and semen sample qualities: a study in 2448 men

Inhibin B is an important serum marker of spermatogenesis, whereas sensitivity and predicting power for the spermatogenic situation at several ages are under debate. We performed a retrospective analysis of data from 2448 men who attended our University-based male infertility clinic to evaluate inhibin B in relation to age and semen sample qualities in comparison with FSH. Moreover, the range of inhibin B in 82 nonobstructive azoospermic patients was correlated with the sperm retrieval in testicular sperm extraction procedures. Inhibin B correlated with FSH (Spearman rank correlation (R)=-0.50; P<0.00001). Inhibin B and inhibin B/FSH ratio (IFR) showed an inverse U-shaped dependence on age, whereas FSH showed a U-shaped dependence on age (optimum 20-40 years). However, in men with normal spermiograms inhibin B concentrations did not differ between age groups. Their levels of inhibin B amounted to 130.5, 54.5-247 ng/l (median, 10th-90th precentile), and of IFR to 38.3, 12.5-104.8 (median, 10th-90th percentile), which might be taken as the reference range. Using the 10th percentile of IFR, correct classification in normal or pathological semen groups was achieved in 99.1%. The percentage of aniline blue-negative spermatozoa, i.e. mature spermatozoa with protamines, did not correlate with FSH (P>0.05) but with inhibin B (R=0.15, P<0.001). The probability of retrieving testicular spermatozoa decreased with declining inhibin B: <20 ng/l sperm could never be found. Our results from a large group of men with a wide spectrum of semen qualities allow estimating reference values for inhibin B and IFR. Inhibin B and especially the IFR are more sensitive markers of male infertility than FSH alone.

Effects of post-density gradient swim-up on apoptosis signalling in human spermatozoa.

The inclusion of apoptotic spermatozoa during assisted reproductive techniques (ART) may be one reason for suboptimal success rates. The aim of our study was to evaluate the potential of routine semen preparation to eliminate spermatozoa with activated apoptosis signalling. Semen samples from 20 infertility patients scheduled for ART procedures were investigated. Following density gradient centrifugation (DGC) and swim‐up, aliquots were taken from each sample to analyse motility, Caspase‐3 activation (CP3) and integrity of the mitochondrial membrane potential (MMP) using flow cytometry. Aliquots from the neat semen served as controls. Semen samples of patients contained 53.8 ± 17.7% spermatozoa with disrupted MMP and 51.8 ± 14.9% with active CP3. Preparation by DGC and swim‐up resulted in improvement of progressive motility (+43.5%) and reduction of spermatozoa with disrupted MMP (−34.3%) and activated CP3 (−25.7%, P < 0.01). Minimal reduction of spermatozoa with disrupted MMP and active CP3 was 6.0% and 0.7%, maximum reduction was 65.5% (disrupted MMP) and 49.3% (CP3). Semen samples of subfertile patients contain high levels of spermatozoa with activated apoptosis signalling. Although there was a reduction in the majority of the samples, profound interindividual differences in the separation effect demand further development of innovative molecular‐based separation methods to deplete apoptotic spermatozoa.