Uwe Paasch

Cryopreservation and Thawing Is Associated with Varying Extent of Activation of Apoptotic Machinery in Subsets of Ejaculated Human Spermatozoa

Abstract We investigated the impact of cryopreservation and thawing on levels of caspases-3, -8, and -9 activity, intact mitochondrial membrane potential (Δψm), and DNA fragmentation in human spermatozoa. Eleven pools of cryopreserved and eight pools of fresh semen samples were examined. Mature and immature fractions were separated on a two-layer density gradient (47% and 90%) and further subdivided based on the externalization of phosphatidylserine and its binding to annexin V-labeled superparamagnetic microbeads (ANMB). Levels of activated caspases were assessed using fluorescein-labeled inhibitors of caspases (FLICA), Δψm using a lipophilic cationic dye, and DNA fragmentation by the terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assay. Cryopreservation was significantly associated with activation of caspases-3, -8, and -9, as well as disruption of the mitochondrial membrane potential but no significant changes were observed in DNA fragmentation. In mature sperm, caspase activation was only detected in the ANMB+ fraction, whereas in immature sperm, both ANMB+ and ANMB− fractions showed activated caspase levels. In ANMB+ immature sperm, apoptosis seemed to be triggered by a surface ligand-receptor mechanism as well as by disruption of mitochondria, whereas in ANMB− immature sperm, apoptosis was induced by activation of caspase-9 following loss of intact Δψm. These results demonstrate that selection of annexin V-negative mature spermatozoa might be of clinical relevance for fertility preservation, as this sperm fraction shows no activated apoptosis during the cryopreservation process.

Selection of Nonapoptotic Spermatozoa As a New Tool for Enhancing Assisted Reproduction Outcomes: An In Vitro Model

Abstract Magnetic cell sorting (MACS) using annexin V-conjugated microbeads eliminates apoptotic spermatozoa based on the externalization of phosphatidylserine residues. The procedure delivers two sperm fractions: annexin V-negative (nonapoptotic) and annexin V-positive (apoptotic). Our aim was to determine whether the sperm fertilizing potential can be improved by selecting a nonapoptotic fraction using MACS. Semen samples (n = 35) were subjected to separation on a density gradient followed by MACS. Extent of apoptosis was assessed by measuring levels of activated caspase 3 using fluorescein-labeled inhibitors of caspase, alterations in mitochondrial membrane potential (MMP) using a lipophilic cationic dye, and DNA fragmentation using terminal deoxynucleotidyl transferase-mediated fluorescein-dUTP nick end labeling assay. The sperm fertilization potential was assessed using hamster oocyte penetration assay and hamster oocyte-intracytoplasmic sperm injection (ICSI). Annexin V-negative sperm displayed superior quality in terms of high motility, low caspase 3 activation, MMP integrity, and small extent of DNA fragmentation. Annexin V-negative sperm demonstrated higher oocyte penetration capacity but comparable sperm chromatin decondensation (SCD) following ICSI. Conversely, the annexin V-positive sperm presented with poor quality and fertilization potential. The oocyte penetration rate was negatively correlated with apoptotic marker expression, whereas SCD following ICSI was only associated with apoptosis on sperm-damaged membranes. We conclude that apoptosis appears to impact sperm-oocyte penetration rate; however, it does not seem to affect early stages of fertilization such as SCD in spermatozoa of healthy donors. The selection of nonapoptotic sperm by MACS may be used to enhance results of in vitro fertilization by increasing sperm-oocyte penetration.

Mature human spermatozoa do not transcribe novel RNA

Mature spermatozoa contain subsets of mRNA that have been found in human testes before. Based on this finding it was hypothesized that the mRNAs of spermatozoa are transcribed during spermatogenesis. However, up to now there is no proof of the transcriptional inactivity of human sperm. To address this issue we performed in vitro labelling experiments with radio‐labelled uridine triphosphate followed by analysis of cellular RNA. There was virtually no radioactive RNA detectable in the RNA purified from human spermatozoa proving the transcriptional inactivity of mature spermatozoa. The spermatozoal RNA obviously results from transcription during spermatogenesis and can be used for diagnostic purposes. These findings might have diagnostic and – possibly – therapeutic value in infertility patients as spermatozoal RNAs might complement the RNA pool of the oocyte after fertilization.

Advantage of combining magnetic cell separation with sperm preparation techniques

The selection of vital, non-apoptotic spermatozoa is a prerequisite for achieving optimal conception rates in assisted reproductive techniques. Magnetic cell sorting using annexin-V microbeads can effectively separate apoptotic and non-apoptotic spermatozoa. The objective of the present study was to optimize the integration of magnetic cell sorting in standard sperm preparations and to correlate the effect of different sperm preparation procedures on apoptotic markers. Semen specimens collected from 15 healthy donors were prepared by either density gradient centrifugation or by one-step sperm wash technique separately and in combination with magnetic cell sorting. The preparation methods were evaluated by assessment of semen parameters (motility, viability and morphology) as well as markers of apoptosis (levels of active caspase-3, integrity of membrane mitochondrial potential and externalization of phosphatidylserine). The apoptotic markers were measured using fluorochrome dyes coupled with flow cytometry. The results showed that the combination of density gradient centrifugation and annexin-V magnetic cell sorting was superior to all other sperm preparation methods in terms of providing motile, viable and non-apoptotic spermatozoa. This study clearly shows the advantage of integrating magnetic cell sorting as a part of sperm preparation, which in turn may positively affect the success rates of assisted reproductive techniques.

Enrichment of non–apoptotic human spermatozoa after cryopreservation by immunomagnetic cell sorting

Cryopreservation increases the rate of apoptotic spermatozoa withdecreased capability to fertilise oocytes. In order to optimise thefertilisation rates, especially in assisted reproduction the use of apoptoticsperms should be avoided. Early events of apoptosis in cryopreservedspermatozoaare not detectable by conventional methods. However, the surface of apoptoticspermatozoa is characterised by externalisation of phosphatidylserine (PS),which has a high affinity to Annexin V. Therefore, colloid paramagneticAnnexin-V-conjugated microbeads (AN-MB) were tested fortheir ability to eliminate apoptotic spermatozoa from a total of 40 fresh andinTEST yolk buffer cryopreserved semen samples which were provided by 15 healthyvolunteers. By passing through a magnetic field (MiniMACS, Miltenyi Biotec) thesperm suspensions were divided into 2 sperm fractions depending on boundmagnetic Annexin V-microbeads (AN-MB) to spermatozoa. Asadditional markers of apoptosis CD95 (Fas, APO-1) on the sperm surfaceand activated caspases in the cytosol were detected in both fractions.Supplementary investigations comprised eosin-supravital staining andcomputer assisted sperm motion analysis. The separation was supervised by flowcytometric analysis of spermatozoa labelled with FITC-conjugated antiAnnexin V-antibodies.Analyses of the magnetic inactive sperm fraction (AN-MB-negative)showed CD95 on 0.6 ± 0.3% (X ± SEM) of spermatozoa andonly3.2 ± 0.5% were stainable with eosin, whereas, 40.6 ±6.7% of the remaining cells in the column appeared to be CD95 positiveand 99.8 ± 0.1% stainable with eosin after cryopreservation.Indeed the overall amount of CD95 positive spermatozoa did not significantlyincrease after cryopreservation (2.5 ± 0.5% vs. 4.3 ±1.2%; p > 0.05). Activated caspases were found in 21.8 ±2.6% of the spermatozoa in fresh and in 47.7 ± 5.8% ofcryopreserved semen samples (p < 0.01). The separation procedure of thecryopreserved spermatozoa reduced significantly the quantity of thosecontainingactivated caspases to 9.3 ± 2.2% within theAN-MB-negative fraction. In contrast 89.1 ± 2.3% ofAN-MB-positive sperms showed activation of these proteolyticenzymes. Flow cytometric analyses using FITC-conjugated anti AnnexinV-antibodies for monitoring of AN-MB-binding to spermatozoashowed 5.2 ± 1.0% labelled spermatozoa in the AN-MBnegative fraction and 72.6 ± 2.7% labelled spermatozoa in theAN-MB positive one. There was no significant influence of the separationcolumn and the magnetic field on the sperm functions. The passage through thecolumn led to a sperm loss of 0.8 ± 1.2%.Conclusion: The binding of paramagnetic AnnexinV-conjugated microbeads is an excellent method to eliminate spermatozoaat early apoptotic stages from cryopreserved semen samples. A deleteriousinfluence of the separation column and the magnetic field on the spermatozoawasnot observed.

Semen quality in sub‐fertile range for a significant proportion of young men from the general German population: a co‐ordinated, controlled study of 791 men from Hamburg and Leipzig

Population studies have shown that a high proportion of Nordic men may have so poor semen quality that they can be classified as sub-fertile according to international standards. A question is whether the Nordic data are specific for the Nordic countries or they should be seen as an expression of a general trend in Europe. We therefore carried out a prospective study of semen quality of young men raised in the former East Germany (Leipzig) and West Germany (Hamburg). To enable inter-regional comparisons, we utilized a common European research protocol previously used in studies in the Nordic-Baltic region. Three hundred and thirty-four young men representative of the general population from Hamburg, and 457 from Leipzig delivered semen samples, underwent physical examinations and provided information on life-style and reproductive health parameters. The study period in Hamburg was February 2003--July 2004, and in Leipzig July 2003--April 2005. No significant differences were observed in sperm concentration (median 46, 42, and 44 million/mL for men from Hamburg, Leipzig and the combined Hamburg-Leipzig group respectively) or total sperm count (154,141 and 149 million), whereas the differences for morphologically normal spermatozoa (9.4 and 8.4%) and motile spermatozoa (67 and 81%) were significantly different. Previously published studies have shown reduced fertility with decreasing sperm concentrations below 40-55 millions/mL and normal sperm morphology below 9-19%. Thus, a large fraction of young German men seem to have impaired semen quality that may reduce their natural fertility. However, it remains to be investigated to what extent poor semen quality contributes to the low German fertility rates.

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.

Leptin exists in tubuli seminiferi and in seminal plasma

Summary.  Leptin is a 167‐amino acid protein that stimulates gonadotrophin‐releasing hormone secretion and exerts indirect effects on the gonads via neuropeptide Y, NPY. Recent research has suggested that leptin may also have an effect on testosterone secretion. To investigate the role of leptin in reproduction, leptin in testicular tissue and seminal plasma was examined in relation to leptin in serum, semen sample qualities and vasectomy. Seminal plasma and serum of 64 infertility patients, and 15 individuals after vasectomy, were assayed for leptin using a competitive ‘in house’ radioimmunoassay. The concentration of leptin in seminal plasma was significantly lower in the ‘normal’ semen sample group than in the ‘pathological’ group (Mean ± SEM; 1.45 ± 0.18 vs. 3.19 ± 0.57 ng ml−1; P < 0.05), and showed a significantly negative correlation with percentage of motile spermatozoa (r=−0.46; P=0.0005) and with the velocity straight line, VSL, (r=−0.30; P= 0.029). In contrast, leptin concentration in serum did not show any relationship with the spermiogram parameters. In testicular tissue, leptin was preferentially found within the tubuli seminiferi using anti‐leptin polyclonal antibody, Ob A‐20 Sc 842. The amount of leptin per ejaculate did not significantly change after vasectomy, and was not correlated to fructose, zinc or neutral alpha glucosidase in seminal plasma (P > 0.05). These results suggest that the amount of leptin in the genital tract, including the tubuli seminiferi, may influence the mechanisms involved in the motility development of spermatozoa.

Deterioration of spermatozoal plasma membrane is associated with an increase of sperm lyso-phosphatidylcholines

Summary.  Spermatozoa with plasma membranes that lost their asymmetry or permeability for larger molecules can be identified by binding of annexin V to membrane phosphatidylserine (PS). Paramagnetic annexin‐V‐conjugated microbeads (AN‐MB) can be used to eliminate these spermatozoa by magnetic activated cell sorting (MACS). Semen samples of six healthy volunteers with normal spermiogram parameters were divided into two sperm fractions by MACS as a function of bound AN‐MB, and their individual lipid compositions were examined by matrix‐assisted laser desorption and ionisation time‐of‐flight mass spectrometry (MALDI‐TOF MS). As a model system, liposomes composed of phosphatidylcholines (PC) from egg yolk were digested by phospholipase A2 (PLA2). The MALDI‐TOF mass spectra of organic extracts of both sperm subpopulations differed significantly. The ratio between lyso‐phosphatidylcholine LPC 16 : 0 and PC 16 : 0/22 : 6 was approximately 2.5–4.7‐fold higher (median 2.9) in the sperm group binding AN‐MB than in spermatozoa with intact membrane unable to bind AN‐MB. The ratio between LPC 22 : 6 and PC 16 : 0/22 : 6 was also enhanced in the spermatozoa with impaired membrane structure (factor in the range: 1.9–3.9; median 2.6). These alterations corresponded to the effects of PLA2 on artificial phospholipids. It is concluded that spermatozoa with deteriorated membrane and exposed PS are characterized by an increased lyso‐phosphatidylcholine content that is likely generated by phospholipases.

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.