Case report: Changes in motility patterns during in-vitro culture of fresh and frozen/thawed testicular and epididymal spermatozoa: implications for planning treatment by intracytoplasmic sperm injection

The present report describes the motility changes in vitro (percentagemotile and progressively motile) of freshly collected testicularand epididymal spermatozoa and following freeze/thaw of thesame spermatozoa from a man with obstructive azoospermia. Washedspermatozoa were cultured in micro droplets under paraffin oilor in test tubes using HEPES-buffered or bicarbonate-bufferedmedium containing 10% human serum. In fresh testicular spermcultures 60–65% of the sperm cells became motile within2 days of culture; the motility was maintained for a further4–5 days before a decline was observed. The progressivemotility unproved markedly on the third day of culture and itpeaked around day 5. Only a small number of frozen/ thawed testicularspermatozoa became motile during in-vitro culture (15–20%)and the motility was maintained for only 2–3 days beforeit declined. Furthermore, only 10–12% of the spermatozoashowed progressive motility. Spermatozoa recovered from micro-epididymalsperm aspiration (MESA) showed a gradual decrease in progressivemotility and in 5 days all sperm cells were found to be immotilein both freshly collected and frozen/thawed spermatozoa. Allculture systems supported sperm motility. It is clear that testicularspermatozoa, particularly from men with obstructive azoospermia,can be collected and maintained in vitro for up to 1 week beforethe oocyte retrieval but when frozen testicular or epididymalspermatozoa are used it is more reliable to thaw these spermatozoaon the day of intracytoplasmic sperm injection.     

Andrology: Density adjustment of software settings minimizes bias in automated sperm motility estimation

To minimize overestimation of motility, it is recommended thatfresh semen be diluted with seminal plasma prior to automatedanalysis. However, for glycerolated or cryo-preserved sementhis is impractical, and alternative methods are needed to minimizeautomated motility bias. In the present study, the proportionof motile spermatozoa was determined in fresh, diluted and cryopreservedsemen (n = 25 ejaculates) using visual and automated methods.The effect of software settings on motility was investigatedby assessing samples at a range of modified settings. At standardsettings, automated motility was biased in fresh semen (+7.2%)after dilution with cryopreservative (–2.9%) and aftercryopreservation (–7.8%) (P < 0.0001 versus visual).Automated motility was inversely related to the minimum numberof frames for motility sampling (P < 0.0001), with mean estimatesof 41.0, 46.1, 52.0 and 58.2% generated at settings of 8, 4,2 and 1 frame(s) respectively (n = 15 fresh, diluted and cryopreservedsamples). Based on an arbitrary ordinal scale, a method wasdeveloped whereby motility sampling was adjusted prior to analysisaccording to sperm density. Analysis of an independent set ofsemen samples with density-adjusted software settings reducedbias in automated estimates (n = 30) before and after freezing(P < 0.0001). In addition, bias was no longer related tosperm density. In conclusion, modification of software settingsis an effective alternative to dilution to minimize bias inautomated motility estimates in fresh, diluted and cryopreservedhuman semen.     

Andrology: Cystic fibrosis mutations impair the fertilization rate of epididymal sperm from men with congenital absence of the vas deferens

One of the limiting factors for the successful treatment ofmale sterility due to congenital absence of the vas deferens(CAVD) is the low (<20%) and extremely unpredictable rateof in-vitro fertilization of their epididymal spermatozoa. Therecent demonstration that CAVD is a mild form of cystic fibrosis(CF) disease, almost exclusively involving the genital tract,has prompted us to investigate the hypothesis of whether therecould be an association between particular CF genotype mutationsand the in-vitro performance of epididymal sperm. In this study63 patients with surgically confirmed diagnosis of CAVD undergoingmicrosurgical epididymal sperm aspiration (MESA) and in-vitrofertilization (IVF) were evaluated. The genetic screening wascarried out on DNA extracted from peripheral lymphocytes andamplified by the polymerase chain reaction. A total of 12 mutationsin the cystic fibrosis transmembrane regulator gene (CFTR),representing 90% of the total known CF mutations, were tested.The presence or absence of mutations, as well as the type ofmutation found, was correlated with the IVF and pregnancy rates.Of the 63 patients examined, 40 (64%) were positive to the CFscreening and 23 were negative. In three cases no spermatozoawere found because of rete testis blockage. The difference infertilization rates between patients testing positive and negativewas highly significant (P = 0.000003), 13 versus 23%, respectively.In order to pinpoint which mutation could account for the mostdeleterious effect on the IVF results, we analysed these bykeeping the patients separated by CF mutations. Three majorgroups were identified: group I was formed by 21 patients (52%of the positive) with Delta F508, the most common and lethalof the CF mutations, as the only detectable. Group II was formedby 18 patients with various other CF mutations, including fourcompound heterozygotes (three with Delta F508/R117H and onewith R553X/R117H) and one homozygote (R117H/R117H). Group IIIwas formed by 21 patients in whom no mutations could be detected.The results showed a very low fertilization and pregnancy rateper patient (7 and 10%, respectively), in group I, while ingroups II and III the fertilization and pregnancy rates weresimilar (group II, 21 and 39%, respectively; group III, 23 and48%, respectively). The differences in the fertilization andpregnancy rates between groups I and III were statisticallysignificant (P < 0.000001 and P < 0.01, respectively),while there was no difference between groups II and III. Inconclusion, this study demonstrates a correlation between CFmutations and IVF capacity of epididymal spermatozoa retrievedfrom men with CAVD. Men with Delta F508 not opposed to knownCF mutations give the poorest IVF outcomes; also at repeatedattempts their pattern of poor fertilization is maintained,perhaps reflecting sperm intrinsic biochemical defects (alterationof chloride channels). These data open a new avenue in the interpretationof the IVF results from men with CAVD.     

Andrology: Epididymal sperm aspiration with assisted reproductive techniques: difference between congenital and acquired obstructive azoospermia?

From July 1992 to May 1994, 31 cases of obstructive azoospermia-inducedinfertility underwent 35 epididymal sperm aspiration proceduresand assisted reproductive techniques. These included in-vitrofertilization (IVF), zygote intra-Fallopian transfer (ZIFT),subzonal insemination (SUZI) and intracytoplasmic sperm injection(ICSI) with embryo transfer or tubal embryo transfer. The motilespermatozoa were retrieved using a gauge 24 Medicut cannulaand flush medium. Total oocyte fertilization rate was 23.4%and the clinical pregnancy rate was 12.5% (four out of 32 treatmentcycles). Congenital absence of the vas deferens was found in16 cases (51.6%) and secondary genital duct obstruction waspresent in 15 cases (48.4%). In all, 29 aspirations were retrievedfrom the caput of the epididymis and six from the epididymalbody or tail. The fertilization rate for the caput spermatozoawas much less than that for other areas of the epididymis (P< 0.05). Though there were no predictable differences infertilization rates between the congenital and acquired groups,by using epididymal spermatozoa for assisted reproduction, thecongenital group seemed to have a stronger tendency to achievepregnancy (20 versus 5.9%).     

Andrology: Fertilizing ability of immotile spermatozoa after intracytoplasmic sperm injection

Sometimes spermatozoa from ejaculate, epididymis or testis showa total absence of motility. For some patients, however, veryfew spermatozoa with very poor motility can be found after severalhours of incubation (initially immotile spermatozoa). Othersamples show no motility at all even after extended culture(totally immotile spermatozoa). Intracytoplasmic sperm injection(ICSI) is the only method available to select and retrieve asingle immotile or initially immotile spermatozoon and injectit into the oocyte. A total of 103 patients with asthenozoospermiaunderwent ICSI in this study. It was shown that initially immotileand totally immotile spermatozoa, whatever their origin, havethe capacity to fertilize an oocyte after ICSI. No significantdifference could be observed between the fertilizing capacityof testicular or epididymal spermatozoa. Totally immotile ejaculatedspermatozoa, however, fertilized significantly fewer oocytesafter ICSI when compared with initially immotile ejaculatedspermatozoa. Embryos of lower quality tended to be producedwhen totally immotile spermatozoa of any origin were used, comparedwith embryos resulting from initially immotile spermatozoa.Ongoing pregnancies were conceived after ICSI with initiallyimmotile spermatozoa from any origin and totally immotile spermatozoaretrieved from testis only. One biochemical pregnancy was theresult of embryo transfer after ICSI with totally immotile ejaculatedspermatozoa. No supernumerary embryos could be cryo-preservedfor patients with totally immotile spermatozoa from ejaculateor epididymis. For a Kartagener patient, subzonal insemination(SUZI) seemed to be a better approach for obtaining fertilizationand pregnancy than ICSI because no fertilization occurred afterICSI on sibling oocytes. Hence a healthy pregnancy was obtainedafter SUZI.     

Fertilization and early embryology: Ongoing pregnancies and birth after intracytoplasmic sperm injection with frozen--thawed epididymal spermatozoa

In seven patients who did not become pregnant following microsurgicalepididymal sperm aspiration (MESA) and intracytoplasmic sperminjection (ICSI), a subsequent ICSI was performed using previouslycryopreserved supernumerary epididymal spermatozoa without re-operatingon the husband. During the original MESA procedure a mean spermconcentration of 12.3x10⁶/ml was achieved. The supernumeraryspermatozoa were cryopreserved for later use. After thawingfrozen epididymal spermatozoa a mean concentration of 1.9x10⁶spermatozoa/ml was obtained in straws containing a total volumeof sperm suspension of 250 µl. From 68 intact oocytesinjected with frozen—thawed epididymal spermatozoa, atwo pronuclear fertilization rate of 45% and a cleavage rateof 82% were obtained. A total of 17 embryos were replaced inthe seven patients, resulting in two ongoing singleton pregnanciesand one twin delivery. Six embryos were cryopreserved. In conclusion,it would appear mandatory to cryopreserve supernumerary spermatozoaduring a MESA in order to avoid subsequent further scrotal surgery.     

Genetics: The use of epididymal and testicular spermatozoa for intracytoplasmic sperm injection: the genetic implications for male infertility

The results and rationale of using testicular and epididymalspermatozoa with intracytoplasmic sperm injection (ICSI) forsevere cases of male infertility are reviewed. A total of 72consecutive microsurgical epididymal sperm aspiration (MESA)cases were performed for congenital absence of the vas (CAV)and for irreparable obstructive azoospermia. ICSI was used toobtain normal embryos for transfer and fertilization in 90%of the cases. The overall fertilization rate was 46% with anormal cleavage rate of 68%. The pregnancy and delivery ratesper transfer were 58 and 37% respectively. The delivery rateper cycle was 33%. In many cases, no epididymal spermatozoawere available and so testicular sperm extraction (TESE) wasused for sperm retrieval. The transfer rate was lower with TESE(84 versus 96%) and the spermatozoa could not be frozen andsaved for use in future cycles. However, there was little differencein pregnancy rates using epidiymal or testicular spermatozoa.The results were not affected by whether the obstruction wascaused by CAV or failed vasoepididymostomy. Both fresh and frozenspermatozoa gave similar results; the only significant factorappeared to be the age of the female. Because of the consistentlygood results obtained using epididymal sperm with ICSI whencompared with conventional IVF, and the similarly good resultswith testicular tissue spermatozoa, ICSI is mandatory for allfuture MESA patients. All CAV patients and their partners shouldbe offered genetic screening for cystic fibrosis; hence pre-implantationembryo diagnosis should be available in any full service MESAprogramme. It is now clear that even with non-obstructive azoospermia,e.g. Sertoli-cell only, or maturation arrest, there are usuallysome small foci of spermatogenesis which allow TESE with ICSIto be carried out. This means that even in men with azoospermiadue to absence of spermatogenesis or to a block in meiosis,there are usually a few spermatozoa available in the testesthat are adequate for successful ICSI. Finally, it is likelythat some forms of severe male factor infertility are geneticallytransmitted and although ICSI offspring have been shown to becompletely normal, it is possible that the sons of these infertilecouples will also require ICSI when they grow up and wish tohave a family.     

Andrology: Analysis of sperm movement in relation to the oxidative stress created by leukocytes in washed sperm preparations and seminal plasma

The addition of luminol to unprocessed semen samples resultedin the generation of chemiluminescent signals, the intensityof which was highly correlated with the level of leukocyte contamination.Despite the spontaneous oxidant-generating capacity of seminalleukocytes, no correlations were observed between leukocytecontamination and the fertility status of the subjects or anyaspect of the semen profile, including the motility of the spermatozoaor their performance in a hyaluronate penetration assay. Luminol-dependentchemiluminescence and leukocyte contamination were also correlatedin washed sperm suspensions prepared either by repeated centrifugationor on discontinuous Percoll gradients. However, in such spermsuspensions, the spontaneous generation of oxidants by contaminatingleukocytes (>2x10⁴ leukocytes/ml) was invariably associatedwith a decreased capacity for movement. Moreover, causativeassociations between leukocyte contamination, reactive oxygenspecies generation, lipid peroxidation and impaired sperm motilitywere revealed by experiments involving the selective additionor removal of activated leukocytes. From these observationswe can conclude that low concentrations of leukocytes are acommon feature of the human ejaculate and can impair sperm function,particularly in the absence of seminal plasma. These findingshave implications for our understanding of the importance ofleukocytospermia in defining the fertility of human spermatozoain vivo and in vitro.     

Mast cell-sperm interaction: evidence for tryptase and proteinase-activated receptors in the regulation of sperm motility

BACKGROUND: The detection of significant levels of tryptase in human seminal plasma and follicular fluid and of tryptase-positive mast cells (MCs) in the wall of human Fallopian tubes lead us to hypothesize that tryptase may exert regulatory actions on human spermatozoa. METHODS AND RESULTS: Immunoelectronmicroscopy revealed proteinase-activated receptor 2 (PAR-2) in the membranes of the acrosomal region and midpiece of human spermatozoa. These PAR-2 were functional, as exposure of spermatozoa from healthy men (n = 12) with regular standard semen parameters to human recombinant tryptase significantly decreased motility in a dose- and time-dependent fashion. Motile spermatozoa (WHO a + b) were significantly decreased within 10 min of incubation with 1.000 ng/ml tryptase (P = 0.045). After 30 and 60 min, significant reduction of motility was also observed in the presence of lower tryptase concentrations (100 ng/ml, P = 0.037; 10 ng/ml, P = 0.046). The inhibitory effects of tryptase progressed throughout an observation period of 180 min. Furthermore, tryptase effects were reversible after washing procedures and could be inhibited by pretreatment with anti-tryptase antibody or anti-PAR-2 antiserum. CONCLUSIONS: The observations presented raise the possibility that tryptase directly interacts with human spermatozoa during their migration through the female genital tract. Genital tract MCs and their products may be as yet unrecognized factors involved in human fertility/sterility.     

Andrology: The variable effects of 2'-deoxyadenosine on human sperm motility and hyperactivation in vitro

The response of human sperm motility and hyperactivation tothe stimulant 2'-deoxyadenosine (2'-DEA) was studied in vitrousing computer-assisted sperm motion analysis. A total of 20randomly selected individuals with normal sperm counts as definedby the World Health Organization were chosen and their migration-separatedspermatozoa exposed to a range (0.1–10.0 mM) of concentrationsof 2'DEA. The straight line velocity (VSL) was increased abovecontrol values only at 0.1 mM, while the curvilinear velocity(VCL) and lateral head displacement (ALH) were increased significantlyat all concentrations. Linearity of progression (LIN), on theother hand, declined with increasing concentration of 2'-DEA.These changes were related to a significant increase in thenumber of spermatozoa exhibiting hyperactive-like motion. Therewas, however, considerable intra-individual variability in theresponse to 2'-DEA. In some individuals VCL and ALH exhibitedlittle or no response to 2'-DEA, whilst in others an increaseabove the control of 50–55% occurred. The maximum responsefor VCL and ALH occurred at 2.5 mM 2'-DEA. Individuals showedgreater variability in the percentage of spermatozoa exhibitinghyperactivity in response to 2'-DEA, with increases rangingfrom 76 to 948% of the control value, although the maximum responsewas also most commonly seen at 2.5 mM 2'-DEA. The diversityof response to 2'-DEA emphasizes the importance of tailoringdoses to the individual rather than employing one concentrationfor all. Further tests on a subgroup of the individuals examinedthe longevity of spermatozoa in response to 24 h of continuedexposure to 2'-DEA. Prolonged exposure to 0.1 mM 2'-DEA continuedto enhance VSL, while higher concentrations produced detrimentaleffects on all other motion characteristics including hyperactivation.     

Andrology: Evaluation of a computer-aided semen analysis system with sperm tail detection

The aim of this study was to evaluate the Stroemberg-Mika cellmotion analyser (SM-CMA) which uses tail detection in orderto discriminate between immotile spermatozoa and other particles.Analysis of the spermatozoa by the SM-CMA can easily be checkedon a video monitor. The semen samples were from donors and patientsvisiting the fertility unit of the University Hospital, Hanzeplein,The Netherlands. Both fresh semen samples and purified spermsuspensions were used to estimate sperm counts and motilitycharacteristics. We tested the use of the x10 objective insteadof the x20 and we assessed the ways in which motility characteristcswere influenced by temperature. We found a considerable discrepancybetween sperm concentrations measured manually and by computerizedanalysis, both in semen samples and in purified sperm suspensions.The SM-CMA correctly recognizes motile spermatozoa, but underestimatesimmotile ones. Although temperature affects motility characteristics,in routine measurements the influence of short cooling periods,which are unavoidable, was nil. We found that using the x10objective can be useful, especially at low sperm concentrations.In our opinion, the SM-CMA system is, despite some shortcomingsin its user-interface, a useful and versatile instrument forexamination of human semen samples, with desirable features.     

Andrology: Effects of nitric oxide on human spermatozoa: evidence that nitric oxide decreases sperm motility and induces sperm toxicity

Endogenous nitric oxide (NO) is an important functional mediatorin several physiological systems, including the reproductivesystem. However, when generated in excessive amounts for longperiods, mainly during immunological reactions, NO is cytotoxicand cytostatic for invading microbes, as well as for the cellsgenerating it and the tissues present around it. Since infertilityassociated with urogenital tract infection in males and femalesis also accompanied by reduced sperm motility and viability,it is possible that reduced fertility in these patients is dueto NO-induced sperm toxicity. We therefore evaluated the directeffects of NO, chemically derived from S-nitroso-N-acetylpenicillamine(SNAP, 0.012–0.6 mM) and sodium nitroprusside (SNP, 0.25–2.5mM), on the motility and viability of human spermatozoa. Furthermore,we tested whether inhibition of NO synthesis prevents spermmotility and viability by incubating washed total cells presentin the semen (spermatozoa, round cells) with N-nitro-L-arginine-methyl-ester(L-NAME), a NO synthesis inhibitor. Treatment of purified spermatozoawith SNAP or SNP decreased forward progressive sperm motilityand straight line velocity, and also increased the percentageof immotile spermatozoa in a concentration-dependent manner.Furthermore, the percentage of immotile spermatozoa positivelycorrelated with the percentage of dead spermatozoa. In contrastto freshly prepared SNAP, SNAP preincubated for 48 h had noeffect on the motility and viability of the spermatozoa. Furthermore,as compared to untreated controls, a significantly higher percentageof forward progressive sperm motility as well as viability (P< 0.05) was maintained in washed semen incubated with L-NAME(0.15 mM). Seminal plasma concentrations of nitrite-nitrate(stabile metabolites of NO/10⁶ spermatozoa correlated positively(P < 0.05) with the percentage of immotile spermatozoa. Ourresults suggest that NO can cause sperm toxicity as well asinhibit sperm motility. In conclusion, excessive NO synthesisin response to infection and inflammation could be an importantfactor contributing to functional change of the spermatozoa,leading to their dysfunction and to infertility.     

Duration of sexual abstinence: epididymal and accessory sex gland secretions and their relationship to sperm motility

BACKGROUND: The data on the association between the abstinence period and sperm motility are conflicting. METHODS: Ejaculates from 422 men assessed for infertility were analysed according to the World Health Organization (WHO) guidelines. Seminal plasma neutral alpha-glucosidase (NAG), prostate-specific antigen (PSA), zinc and fructose were measured. Three groups were defined according to the length of sexual abstinence: G2-3 (2-3 days), G4-5 (4-5 days) and G6-7 (6-7 days). RESULTS: The total percentage of progressively motile spermatozoa was significantly higher in G4-5 compared with G2-3 and G6-7 (medians 55 versus 47 and 42%; P=0.039 and P <0.001, respectively). The percentage of spermatozoa with tail defects was significantly higher in G6-7 compared with G2-3 and G4-5 (medians 14 versus 10 and 10%; P=0.011 and P=0.002, respectively). NAG was significantly lower in G2-3 compared with G4-5 and G6-7 (medians 23 versus 34 and 34 mU/ejaculate; P <0.001 and P=0.001, respectively). The same trend was found regarding zinc (medians 6 versus 8 and 8 mumol/ejaculate; P=0.001 and P=0.005, respectively). CONCLUSIONS: Within the time interval recommended by the WHO (2-7 days), the length of the abstinence period is associated with sperm characteristics and should be taken into consideration when interpreting results of semen analysis.     

Endeometriosis: The influence of peritoneal fluid from patients with minimal stage or treated endometriosis on sperm motility parameters using computer-assisted semen analysis

The aim of this study was to determine the influence of peritonealfluid from patients with minimal stage or treated endometriosison sperm motility parameters. Peritoneal fluid aspirated atdiagnostic laparoscopy for unexplained infertility from womenduring the luteal phase of the menstrual cycle (days 20–23)was incubated for 5 h with fresh semen samples obtained frommen of recently proven fertility. Spermatozoa were preparedby a swim-up technique from unprocessed semen. Using computer-assistedsemen analysis (Hamilton-Thorn Research, MA, USA), sperm motilityand motion parameters were observed at 0, 120, 180 and 300 min.Compared with spermatozoa incubated in Earle's balanced saltsolution/human serum albumin, the percentage motility, percentageprogressive motility and progressive velocity of spermatozoaincubated in peritoneal fluid from patients without visibleendometriosis were significantly higher (P< 0.05). Maximaleffect was observed at 3 h and maintained until 5 h. We concludethat in an in-vitro study, in contrast to peritoneal fluid frompatients with minimal stage endometriosis, peritoneal fluidfrom patients with unexplained infertility and no visible endometriosiscan improve sperm motility when compared with culture medium.     

Effect of recombinant human gonadotrophins on human, bovine and murine oocyte meiosis, fertilization and embryonic development in vitro

The response of murine, bovine and human oocytes to pure recombinant preparations of human follicle stimulating hormone (rFSH) and luteinizing hormone (rLH) for meiotic maturation and subsequent developmental competence in vitro were examined in the present experiments. Maturation of immature bovine oocytes to the metaphase II stage was significantly increased by the addition of 1 IU/ml of rFSH in combination with either 1 IU/ml rLH or 10 IU/ml rLH. Similarly, embryonic development to the blastocyst stage was improved in bovine oocytes treated with a 1:10 combination of rFSH:rLH. However, no significant difference was observed in the number of inner cell mass or trophectoderm cells of the resulting blastocysts. Although the increased maturation to metaphase II was not significant, human embryonic developmental competence was improved by maturing oocytes in the presence of a 1:10 ratio of rFSH:rLH as only those oocytes exposed to a 1:10 ratio of rFSH: rLH during maturation showed normal cleavage patterns beyond day 2. In addition, 1 IU/ml rFSH and 1 IU/ml rLH increased the expression of oocyte proteins in human oocytes. The inclusion of recombinant gonadotrophins, either singly or in combination, had no significant effect on the maturation, fertilization or embryonic development of in-vitro matured mouse oocytes. These data provide support for the responsiveness of human and bovine oocytes to gonadotrophins in vitro and the need to consider variations in the relative concentrations for optimization of oocyte developmental competence.     

Protective role of superoxide dismutase in human sperm motility: superoxide dismutase activity and lipid peroxide in human seminal plasma and spermatozoa.

The levels of superoxide dismutase (SOD), a highly specific scavenging enzyme for superoxide anion radicals (O2-), and lipid peroxide produced by oxygen free radicals were measured in human seminal plasma and spermatozoa. Seminal plasma contained 366.8 +/- 20.9 U/ml (mean +/- SE) of SOD activity. SOD activity in human spermatozoa showed a significant correlation to the number of motile spermatozoa, while the activity in seminal plasma did not relate to the sperm concentration or motility. The lipid peroxide concentration in seminal plasma was 6.22 +/- 0.46 nmol/ml and had no significant relationship to sperm concentration or motility. The malondialdehyde (MDA) concentration in spermatozoa was significantly related to the number of immotile spermatozoa. A decrease in the motility of spermatozoa incubated in medium without seminal plasma was observed after 120 min, while the MDA concentration of the spermatozoa increased. Addition of exogenous SOD (400 U/ml) to the sperm suspension significantly decreased this loss of motility and the increase of the MDA concentration. These data suggest a significant role for SOD in sperm motility. It seems that lipid peroxidation of human spermatozoa may cause loss of motility and that SOD may inhibit this lipid peroxidation. These results suggest that SOD may have a possible clinical application in the use of spermatozoa for in-vitro fertilization (IVF) or artificial insemination.     

Characterization of epithelial cell culture from human hydrosalpinges and effects of its conditioned medium on embryo development and sperm motility

BACKGROUND: Recent studies have reported the negative impact of hydrosalpinx on IVF outcome. Toxic effects of hydrosalpinx fluid (HF) have been the main reason for the recommendation of functional surgery, salpingectomy, prior to IVF. The present study characterized hydrosalpinx epithelial cell culture and examined the effects of its conditioned medium (CM) on sperm motility, acrosome reaction and embryo development. METHODS: Normal Fallopian tubes (n = 6) and hydrosalpinges (n = 9) were used to prepare epithelial cell culture and CM. Epithelial cell characterization was confirmed using electron microscopy. Sperm motility and acrosome reaction were determined using computer-aided sperm analysis and acrobead assay respectively and embryo development by mouse embryo development assay. RESULTS: The percentage of human motile sperm incubated in hydrosalpinx CM was significantly different from those in normal Fallopian tube (NFT) CM and modified human tubal fluid medium (hTF) (control) (P < 0.05 at 3 h and P < 0.001 at 5 and 24 h), with alteration in movement characteristic, linearity, 24 h after incubation in hydrosalpinx CM (P < 0.05). However, other sperm movement characteristics remained unchanged. Reduced acrosome reaction and poor mouse embryo development were also observed in hydrosalpinx CM but not in NFT CM and hTF. CONCLUSIONS: The results suggest that hydrosalpinx epithelial cells may be producing a fluid milieu hostile to sperm and early embryo development. The established epithelial cell culture system may provide a model to further investigate the mechanisms underlying the toxic effects of HF on embryo development and the adverse effects on IVF outcomes.     

Microsurgical epididymal sperm aspiration: aspirate analysis and straws available after cryopreservation in patients with non-reconstructable obstructive azoospermia. MESA/TESE Group Giessen

Microsurgical epididymal sperm aspiration (MESA) combined with intracytoplasmic sperm injection (ICSI) represents a great advance in the therapy of non-reconstructable obstructive azoospermia. For procedure synchronization, a great number of organizational facilities are needed. Intentional cryopreservation of the aspirate may reduce these problems, therefore the aim of this study was to analyse the amount and quality of aspirate fluid obtained by means of MESA and the quality of the vials after thawing. Furthermore, the available cryopreserved straws were calculated. A total of 93 consecutive MESA procedures were performed and epididymal spermatozoa were obtained in 88 patients. Mean sperm concentration was 40.9 x 10(6) spermatozoa/ml. Global and progressive motility were 24.8 and 7.5% respectively. In one-third of the aspirates, no progressive motile spermatozoa were found. The mean number of straws available was 7.6. In 33 ICSI cycles with frozen-thawed epididymal spermatozoa, a pregnancy rate of 42.4% was achieved. In conclusion, these data show that enough spermatozoa are available for various ICSI cycles following a single MESA procedure in men with non-reconstructable obstructive azoospermia. Furthermore, ICSI with cryopreserved spermatozoa leads to excellent pregnancy rates     

Fertilization, embryonic development and implantation of mouse oocytes with one or two laser-drilled holes in the zona, and inseminated at different sperm concentrations

The zonae pellucidae of mouse oocytes were photo-ablated byan ultraviolet laser (337.1 nm) to create one or two 10 µmholes. The pulse energy used was 3 µJ/s, with a frequencyof 10 pulses/s. These laser zona-drilled (LZD) oocytes withone hole (LZD1) or two holes (LZD2) and the zona-intact controlswere inseminated with spermatozoa at standard concentrationsof 5x104, 5x105, 1x106 and 2xl06/ml. Fertilization was significantlyimproved at all sperm concentrations in LZD1 and LZD2 oocytesas compared to the controls. However, there was no significantdifference in fertilization rates between LZD1 and LZD2. LZD2produced significantly higher numbers of blastocysts at day5. Hatching was significantly enhanced in the presence of eitherone or two holes in the zona. Poly-ploidy was generally absent,except in LZD2 oocytes (1%) inseminated at higher sperm concentrations.Differential cell counts of expanded LZD blastocysts were similarto those of the controls. Significantly fewer LZD2 blastocystsimplanted and produced viable fetuses than LZD1 and controlblastocysts. Morphological abnormalities of the fetuses wereabsent in all three groups. Laser zona-drilling using the ultravioletlaser was shown to be fast, efficient and safe.     

CatSper gene expression in postnatal development of mouse testis and in subfertile men with deficient sperm motility

BACKGROUND: The search for Ca2+ channels residing in sperm has led to the recent cloning and characterization of a novel gene, named CatSper, which codes for a unique Ca2+ channel expressed exclusively in the testis. It plays an essential role in sperm motility, penetration into the oocyte, and ultimately in male fertility. In this study, we assessed the temporal profile of CatSper gene expression during mouse testis development and performed a semi-quantitative evaluation of expression levels in a group of subfertile men which lack sperm motility. METHODS: A small piece of testicular tissue obtained by either multi-site testicular biopsy or orchidectomy was used for semi-quantitative RT-PCR of CatSper and beta2-microglobulin (beta2m, as an internal control) genes. RESULTS: Our results reveal that: (i) the expression of mouse CatSper is developmentally regulated with a direct correlation between CatSper expression and mouse sexual maturation. CatSper gene expression is first detected at 3 weeks of age and coincides with the appearance of round spermatids in the developing mouse testis. (ii) There is a significant reduction in the level of CatSper gene expression (up to 3.5-fold difference) among patients which lack sperm motility as compared with patients whose infertility cannot be ascribed to a deficiency in motility (used as a control). CONCLUSIONS: The data obtained in this study support a potential role for CatSper in sperm motility and fertility in mouse and human. CatSper is therefore implicated as a potential target to explore the molecular mechanisms of male infertility.