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.     

The effects of the sperm motility activators 2-deoxyadenosine and pentoxifylline used for sperm micro-injection on mouse and human embryo development

The sperm motility stimulants 2-deoxyadenosine (DOA) and pentoxifylline(PTF), used to improve the success of insemination and spermmicro-injection for low motility sperm samples, were studiedfor their effects on the developmental capacity of mouse andhuman oocytes. When human oocytes were micro-injected with spermatozoain 3 mM DOA 80% of them became blocked at the 1-cell pronuclearstage. However, when spermatozoa in 3 mM PTF were used for micro-injectionor when spermatozoa were washed to remove DOA before micro-injectiononly a few oocytes (9–10%) were blocked. Pregnancies occurredin five of 14 patients into whom cleaving embryos from all threetreatments had been transferred, indicating that once cleavagewas initiated, development of embryos occurred at expected rates.Exposure of mouse oocytes to DOA for a short period during insemination(4–6 h) or a longer period during the pronuclear cellcycle (18 – 20 h) significantly reduced cleavage beyondthe 2-cell stage, resulting in a dramatic reduction in blastocystformation. PTF did not significantly reduce mouse embryo development.Similar results were obtained for oocytes inseminated in vitroor micro-injected with a spermatozoon into the perivitellinespace. Neither DOA nor PTF increased fertilization of mouseoocytes. PTF reduced fertilization, particularly in cumulus-enclosedoocytes and oocytes micro-injected with spermatozoa in PTF.We conclude that DOA is a potent inhibitor of embryo developmentand oocytes should not be exposed to DOA. Exposure of oocytesto PTF had little effect on their subsequent development buttreatment of cauda epididymal mouse spermatozoa can reduce theirfertilizing capacity.     

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: Nuclear decondensation of sperm head and failure at in-vitro fertilization: an ultrastructural study

The problem of unexplained male infertility was investigatedby electron microscopic study of spermatozoa from 51 males.The subjects were subdivided as follows: group A (n = 25) normalfertile males (controls), group B (n = 13) successful in-vitrofertilization (IVF) cases (fertilization rate >50%), groupC (n = 13) failed IVF cases. All subjects included in groupsB and C had a 6–12 year history of childlessness and IVFwas employed when other methods of assisted reproduction failed.The study of spermatozoa in fertile males (controls) was carriedout to establish baseline ultrastructural abnormalities. Inall 51 cases, an average of 330 (280–800) sperm headsand 660 (330–1190) sperm tails were studied. Decondensationof nuclear chromatin was observed in 70 ± 15% (mean ±SD) of spermatozoa in failed IVF cases, 16 ± 5% in successfulIVF cases and 7 ± 3% in controls. These results werefound to be statistically significant (P > 0.001). The meanvalue for motility of spermatozoa in all three groups was withinaccepted limits of normality. It is concluded that decondensationof nuclear chromatin seen by electron microscopy is one of themost important causes of male infertility. It is advocated thatelectron microscopic examination of semen should be carriedout in all cases of longstanding, unexplained male infertilitybefore embarking upon IVF programmes.     

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.     

Pregnancies achieved with testicular and ejaculated spermatozoa in combination with intracytoplasmic sperm injection in men with totally or initially immotile spermatozoa in the ejaculate

The efficacy of intracytoplasmic sperm injection (ICSI) employingtesticular and ejaculated spermatozoa was assessed in 24 coupleswith totally or initially immotile spermatozoa. No criteriawere employed in selecting which patients would be treated withtesticular or ejaculated spermatozoa. The men were chosen atrandom. Testicular spermatozoa obtained by testicular spermextraction were used in 14 and ejaculated spermatozoa were usedin 10 of these couples. In all cases, asthenozoospermia wastotal in their basal semen sample. In 12 male partners, spermatozoawere totally immotile before and after Percoll gradient fractionation(totally immotile). In the remaining 12 men, spermatozoa initiallyshowed a total absence of motility; however, some of the spermatozoahad showed very poor motility (0.1%) after Percoll gradientfractionation and a 13–2.0 h incubation period (initiallyimmotile). Of these 24 total asthenozoospermic males, 14 alsohad total terato-zoospermia. The fertilization and cleavagerates in the testicular and ejaculated sperm groups were 533and 963 and 543 and 94.4% respectively. One cycle resulted incomplete fertilization failure, and in 23 embryo transfer cyclesa total of 10 pregnancies were obtained (41.6%). Eight pregnancieswere achieved in the testicular sperm group, while only twopregnancies were obtained in the ejaculated sperm group. Fourpregnancies, two from the ejaculated sperm group and two fromthe testicular sperm group, resulted in clinical abortions inthe first trimester. Of the remaining six pregnancies, two havealready resulted in healthy births and four pregnancies arenow in the second or third trimester in the testicular spermgroup. Using testicular spermatozoa in combination with ICSIcan be an alternative mode of treatment in cases with totallyor initially immotile spermatozoa in the ejaculate. Very lowpregnancy rates have been obtained and no ongoing pregnancyhas been achieved using ejaculated spermatozoa in these cases.     

Andrology: An auto-controlled study in in-vitro fertilization reveals the benefit of Percoll centrifugation to swim-up in the preparation of poor-quality semen

The efficiency of spermatozoa prepared by swim-up or by Percollcentrifugation was assessed in an in-vitro fertilization programmeon 71 semen samples of a well-defined quality [total numberof type A (WHO criteria) motile spermatozoa]: category I (n= 21) with > 100 x 10⁶, II (n = 31) with 15–100 x 10⁶,III (n = 11) with 5–15 x 10⁶ and IV (n = 8) with <5 x 10⁶ type A motile spermatozoa. Oocytes were inseminated4 h after oocyte retrieval, alternately with spermatozoa derivedfrom swim-up and Percoll preparation. Both selection proceduresresulted in a significantly higher (P < 0.001) percentagemotility as compared to fresh semen. For low-quality samples(III and IV), however, swim-up was more effective in selectinghighly motile (P = 0.004) and morphologically normal spermatozoa(P < 0.05). For high-quality samples, this difference mighthave been masked by introducing a swim-up step to remove Percollparticles. Regardless of the initial sperm quality, the meanfertilization rate was significantly higher (P = 0.003) whenPercoll-treated spermatozoa were used for insemination (51.3versus 37.8%). For semen of groups I and II, no difference infertilization capacity was observed according to the sperm preparationmethod. Despite the lower percentage motility and normal morphologyfor the Percoll compared to the swim-up treatment in groupsIII and IV, fertilizing capacity was significantly (P < 0.001)in favour of this selection method (65.3 versus 26.5% in groupIII, 47.6 versus 11.6% in group IV). Based on these results,it may be concluded that a subgroup of patients exhibiting poorsemen quality can benefit from Percoll semen preparation interms of improved fertilizing capacity.     

Expression analysis of the human testis-specific serine/threonine kinase (TSSK) homologues. A TSSK member is present in the equatorial segment of human sperm

Two members of the human testis-specific serine/threonine (Ser/Thr) kinase family, TSSK 1 and TSSK 2, were cloned and sequenced from a human testis adaptor-ligated cDNA library using a PCR strategy. Within the cDNA, open reading frames (ORF) were defined encoding proteins of 367 and 358 amino acids respectively, as well as conserved kinase domains typical of the superfamily of Ser/Thr kinases. Both genes were intronless and mapped to chromosomes 5 and 22 respectively. The human and mouse homologues of TSSK 1 and TSSK 2, together with TSSK 3 and SSTK/FKSG82, constitute a kinase subfamily closely related to the calmodulin kinases and SNF/nim 1 kinase subfamilies. Similar to the mouse, tissue expression by northern and dot blot analysis revealed that human TSSK 1 and 2 messages are expressed exclusively in the testis. However, mRNA for these kinases can be detected in other tissues using real-time PCR. In addition, TSKS, the human homologue of a putative substrate of TSSK 1 and 2, was cloned. TSKS had an ORF of 592 amino acids and was also expressed exclusively in the testis as demonstrated by northern and dot blot analyses; however, lower levels of expression in other tissues were detected using real-time PCR. Human TSSK 2 and TSKS interacted in a yeast two-hybrid system and also co-immunoprecipitated after in vitro translation. TSSK 2 expressed in yeast and bacteria was able to autophosphorylate and also phosphorylated recombinant TSKS in vitro. Antibodies against recombinant TSSK 2 demonstrated that a member of the TSSK family was present in human testis and localized to the equatorial segment of ejaculated human sperm. In contrast, TSKS was only found in the testis. The finding of a TSSK family member in mature sperm suggests that this family of kinases might play a role in sperm function.     

Induction of capacitation in human spermatozoa in vitro by an endometrial sialic acid-binding protein

A Ca²⁺-dependent sialic acid-binding protein (SABP) of humanendometrium, which specifically bound to human sperm head plasmamembrane in vitro, was found to increase the percentage motilityand acrosome-reacted pattern of uncapacitated spermatozoa. Theprotein was synthesized in the endometrium and secreted intothe uterine fluid. This intra-uterine factor, which is apparentlyadvantageous in vitro in inducing human sperm capacitation,may play a significant role in promoting the postrelease maturationof ejaculated spermatozoa by enhancing ⁴⁵Ca uptake into spermatozoaby a pathway which is insensitive to calcium-channel blockers.However, the ⁴⁵Ca uptake could be enhanced on exposure to thedivalent cation ionophore A23187 and inhibited in the presenceof the calmodulin inhibitor trifluoperazine. The SABP also inducesan increase in intracellular Ca²⁺ in spermatozoa, as seen byFURA-2 AM studies. Furthermore, overlay studies show human SABPto be a Ca²⁺-binding protein. The data presented here suggestthat SABP induces invitro sperm capacitation and the subsequentacrosome reaction by increasing intracellular Ca²⁺ concentration.     

Comparison between chromatin condensation and morphology from testis biopsy extracted and ejaculated spermatozoa and their relationship to ICSI outcome

A significant association between male subfertility, imperfect spermiation and abnormal nuclear condensation has been suggested. The DNA content of spermatozoa might be responsible for inducing alterations in sperm morphology. The final nuclear shape, which is species-specific, depends on chromatin condensation during spermatogenesis as well as a precise organization of DNA within the nucleus. Many reports have described the association between disturbances in sperm chromatin condensation, morphology and male infertility. Chromatin condensation is achieved by gradual substitution of lysinerich somatic histones by testis-specific histone and finally by protamine. In this study two groups of patients were compared: the first consisted of 63 patients who had undergone intracytoplasmic sperm injection (ICSI) with freshly ejaculated spermatozoa whereas the second included 47 patients assigned to ICSI with testes biopsy-extracted spermatozoa. In both groups chromatin condensation was assessed by aniline blue staining and morphology evaluated according to strict criteria. The condensed chromatin and morphology of spermatozoa were significantly (P < 0.0001) less in the second group compared to the first. However the fertilization, cleavage, implantation and pregnancy rates were almost the same in both investigated groups. There was no significant difference between the two groups with respect to ICSI outcome. The percentage of chromatin condensation (nuclear maturity) and morphologically-normal spermatozoa were significantly higher (P < 0.0001) in the ejaculated spermatozoa than in those from testis biopsy but the ICSI outcome (fertilization, cleavage, implantation and pregnancy rates) was the same. In view of these results the fertilization capability and the embryo quality obtained using testis biopsy extracted spermatozoa is not influenced by chromatin condensation and sperm morphology in testicular sperm extraction (TESE)-ICSI programmes. Therefore, it could be said that neither chromatin condensation nor morphology of testis extracted sperm could predict the fertilization, implantation and pregnancy rate in TESE-ICSI programmes.     

Computer image sperm selection as a novel approach to subzonal insemination in the human

Utilizing real-time computer image analysis, individual spermatozoawere selected using microaspiration. Selection criteria werebased on potential hyperactivation motility characteristics;the amplitude of lateral head displacement >7.5 µm,curvilinear velocity >70 µm/s and linearity of <30%.For this pilot study, 16 patients (eight in each group) wererecruited. Using subzonal insemination (SUZI), up to five (mean= 4.4 ± 0.3) spermatozoa selected using computer-imagesperm selection (CISS) were microinjected, or up to 15 (mean= 12.8 ± 1.3 SD) unselected spermatozoa. In the groupwhich utilized CISS, 28 out of 49 (57%) oocytes were fertilizedcompared with 13 out of 52 (25%) utilizing conventional SUZI(P < 0.04); polyspermy was 20% (n = 10) and 2% (n = 1) respectively.CISS with SUZI showed increased efficiency in achieving fertilizationand is a novel approach to studying individual sperm functionin a sperm egg bioassay where gamete ratios are close to unity.     

Andrology: Deleterious effects of khat addiction on semen parameters and sperm ultrastructure

The semen parameters and sperm ultrastructural morphology havebeen described in semen samples from two groups of Yemeni subjects.The first ‘exposed’ group comprised 65 khat addicts,while the second control group included 50 non-khat addict subjects.The mean age was 39.94 ± 13.85 and 35.72 ± 11.35years in the exposed and control groups respectively, withouta significant difference. The mean duration of khat addictionamong the addicts was 25.34 ± 12.96 years (range 6.00–48.00).Statistically significant differences were detected betweenthe semen parameters of the two groups. Such parameters, includingsemen volume, sperm count, sperm motility, motility index andpercentage of normal spermatozoa, were lower among addicts.Significant negative correlation was also found between theduration of khat consumption and all semen parameters (r rangedfrom –0.30 to –0.74). At the transmission electronmicroscopy level, a counting system was incorporated to comparethe numbers of normal spermatozoa with deformed and dead spermatozoain ultrathin plastic sections. The total mean percentage ofdeformed spermatozoa was {small tilde}65%. Different patternsof sperm deformation were demonstrated, and included both thehead and flagella in complete spermatozoa, aflagellate heads,headless flagella and multiple heads and flagella. Deformedheads showed aberrated nuclei with immature nuclear chromatinand polymorphic intranuclear inclusions; these were associatedwith acrosomal defects. The deformed flagella demonstrated numericaberrations of the axonemal 9 + 2 configuration and structuraldefects of their associated elements. Persistent cytoplasmicdroplets were observed frequently. This study has shown forthe first time the deleterious effects of khat addiction onsemen parameters in general and sperm morphology in particularof all addicts, especially those who have consumed khat forlonger periods of time.     

The Acridine Orange test: a clinically relevant screening method for sperm quality during infertility investigation?

To determine the clinical usefulness of Acridine Orange (AO)staining of spermatozoa as a screening test for the evaluationof semen quality during basic infertility investigation, semensmears from 103 randomly chosen males of subfertile coupleswere examined. The median duration of infertility was 4.5 years(range 1–15) and the median age was 33 years (range 21–53).The outcome of AO staining ranged from 5 to 81%, with a medianof 24%, green fluorescent spermatozoa. Results were not significantlyrelated to the parameters of semen analysis (sperm count, motility,standard morphology, viability, pH and volume, as well as fructoseconcentration and number of round cells) or to local sperm antibodytesting and semen cultures. Fluorescence after AO staining wasalso not related to sperm functional capacity (evaluated usingsperm-mucus interaction tests in vitro and in vivo), or themedical history of the patient. No significant differences inthe AO test outcome were seen in patients with explained andunexplained infertility, or with regard to subsequent fertility[with a median value of 21% (range 5–46) green fluorescencein the fertile group, compared with a median value of 28% (range9–81) green fluorescence in the other men]. The resultsof this prospective study indicate that under the usual conditionsof conception, the AO test is not clinically useful as a screeningprocedure to determine semen quality during basic infertilityinvestigation.     

A self-migration method for preparation of sperm for in-vitro fertilization

Human sperm samples (n = 211) were prepared for in-vitro fertilization(IVF) and embryo transfer by a self-migration procedure in Earle'smedium containing highly purified hyaluronic acid (Hya) (MW3 000 000) included to increase the viscosity of the medium.The method resulted in the recovery of a significantly higherpercentage of motile spermatozoa compared with the traditionalcentrifugation method, 87.5 ± 0.9% versus 76.1 ±1.3% (P < 0.001). When comparing media with and without Hyain the selfmigration method for preparation of normal spermsamples, the media containing Hya resulted in the recovery ofa significantly higher percentage of motile spermatozoa, 89.0± 0.8% versus 73.8 ± 2.0% (P < 0.001). In agroup of 80 consecutive couples entering our IVF programme,sperm samples from 44 of the men were allocated at random forthe self migration method in medium containing Hya and spermsamples from 36 men for preparation by centrifugation and swim-up.Significantly more pregnancies were achieved in the group preparedin medium containing Hya. It is concluded that self-migrationof sperm in a medium containing Hya is simple and rapid, andresults in a high recovery of motile spermatozoa which can beused for in-vitro insemination of human oocytes with favourableresults.     

Andrology: Pentoxifylline-supplemented cryoprotectant improves human sperm motility after cryopreservation

In this study, human spermatozoa obtained from donors (n = 15)with normal semen characteristics were cryopreserved in humansperm preservation medium, supplemented with the phosphodiesteraseinhibitor pentoxifylline at concentrations of 0, 1, 3 and 10mM. The effect of pentoxifylline on cryopreserved spermatozoawas determined by monitoring changes in sperm motility and acrosomemorphology by labelling the spermatozoa with fluorescein-conjugatedconcanavalin A lectin. Cryoprotectant supplemented with 1 mMpentoxifylline was found to improve post-thaw progressive motilityfrom 15.3 ± 2.4 (control) to 23.1 ± 3.8% (P <0.01), and total motility from 27.4 ± 3.3 (control) to38.2 ± 3.9% (P < 0.05) without reducing the percentageof spermatozoa with normal acrosomal regions, and so appearsuseful for cryopreservation purposes. The beneficial effectsof 1 mM pentoxifylline on sperm motility were shown to be maintainedpost-thaw over a 6 h time course. Cryoprotectant supplementedwith 3 mM pentoxifylline was found to improve only post-thawprogressive motility, from 15.3 ± 2.4 (control) to 20.7± 3.0% (P < 0.05). However, cryopreservation in thepresence of 10 mM pentoxifylline was found to have a significantly(P < 0.01) detrimental effect on acrosome morphology post-thaw,reducing it from 29.0 ± 2.0 (control) to 21.0 ±2.4% without affecting sperm motility. This suggests that assessmentof the acrosomal region may indicate subtle deleterious effectsof cryoprotectant supplements that cannot be determined frompost-thaw motility assessments alone. These findings differfrom previous studies in that a lower concentration of pentoxifylline(1 mM) was found to be optimal for cryopreservation purposes.     

Sperm selection by PD-10 sephadex columns: comparison with SpermPrep filtration and Percoll centrifugation

The efficacy of a disposable, prepacked column (PD-10) containingSephadex G-25, to select motile spermatozoa, was compared withanother column for sperm filtration (SpermPrep) and centrifugationthrough Percoll gradients. Aliquots of washed sperm suspensionswere processed by the three techniques. The number of motilecells and the proportion of total spermatozoa selected was similarfor all methods. Recovery of spermatozoa showing optimal movementwas 145.9 ± 30% (mean ± SEM) with PD-10 columnsand 131.9 ± 32% with Percoll, both significantly higherthan SpermPrep (71.9 ± 11%; P < 0.05). The straightline velocity of motile cells was lower in samples processedby SpermPrep (29.3 ± 2 µm/s) compared to both PD-10(34.7 ± 1 µm/s) and Percoll (34.9 ± 2 µm/s;P = 0.07). When whole semen was processed, total sperm recoverywith PD-10 was 61.7 ± 8% versus 47.7 ± 7% withPercoll (P < 0.001). Percoll centrifugation improved thepercentage of morphologically normal spermatozoa more than PD-10.Similar proportions of motile spermatozoa and cells with optimalmotility were obtained by both methods. We conclude that PD-10filtration columns can be used to prepare semen in the laboratoryas a practical alternative to other methods.     

Studies of percutaneous epididymal sperm aspiration (PESA) and intracytoplasmic sperm injection

Four distinct studies were carried out using two data sets ofpercutaneous epididymal sperm aspiration (PESA) and intracytoplasmicsperm injection (ICSI) procedures performed from March 1993to January 1997. In study A, an analysis of 181 ICSI treatmentcycles following PESA revealed a successful epididymal spermretrieval rate of 83%. It confirmed that PESA is an effectivesperm retrieval method and the associated ICSI pregnancy rate(35% per embryo transfer) compared favourably with that of othersperm retrieval methods. In study B, the relevance of a priordiagnostic PESA procedure was ascertained by comparing the spermretrieval rates in two groups of patients having their firstICSI treatment cycle with spermatozoa retrieved through PESA.Group B1 (n=50) had diagnostic PESA prior to the ICSI treatmentcycle PESA procedure, unlike patients in group B2 (n=64) whodid not. The sperm retrieval rate in the treatment cycle procedurewas not different at 90 and 82.8% for groups B1 and B2 respectively.However, the discontinuation of diagnostic PESA is fraught withproblems including liability to medico-legal sanctions. In studyC, analysis of 177 treatment cycles involving PESA and ICSIrevealed a successful sperm retrieval rate by PESA of 82% inthe first cycle, 93% in the second, 96% in the third and 100%in the fourth cycle. The same trend was evident when sperm retrievalwas examined in relation to each of the epididymides. Retrievedspermatozoa were found to be motile in 67-100% of cases andthe frequency of samples containing motile spermatozoa did notdecrease with increase in the number of PESA attempts. Theseresults show that PESA does not jeopardize future epididymalsperm retrieval. In study D, the outcome of treatment with ICSIusing ejaculated spermatozoa (305 cycles) (group D1) was comparedwith that of ICSI using spermatozoa obtained through PESA (54cycles) (group D2). The median age of women in the two groupsof couples was similar (34 years). In group D1, 70% of metaphaseII oocytes were fertilized compared with 61% in group D2 (P<0.01).The cleavage rate and the median numbers of transferred andcryopreserved embryos were similar in both groups. There wasno significant difference between the clinical pregnancy rates(33 and 42% in groups D1 and D2 respectively). Our results showthat the outcome of PESA-ICSI treatment compared favourablywith that of ICSI using ejaculated spermatozoa.     

The possible meaning of transferrin and its soluble receptors in seminal plasma as markers of the seminiferous epithelium

Transferrin (Tf) and soluble transferrin receptors (S-Tf-R)were measured by enzyme immunoassay in seminal plasma of 130semen samples. The mean concentration of S-Tf-R in cases withnormozoospermia was 10.4 IU/ml (95% confidence interval: 9.5–11.3)and it was significantly lower in patients with oligozoospermia(6.6, 95% CI: 5.8–7.5, P < 0.001), asthenozoospermia(8.5, 95% CI: 5.5–10.7, P < 0.05), azoospermia of primarytesticular origin (7.9, 95% CI: 6.1–9.6, P < 0.05)and post-vasectomy samples (5.9, 95% CI: 5.4–6.9, P<0.001). The concentration of S-Tf-R in post-vasectomy sampleswas lower than that in patients with azoospermia of primarytesticular origin (P< 0.05; positive likelihood ratio= 7at value of 8.3 IU/ml). S-Tf-R was positively correlated withmotile sperm concentration (r= 0.50, P< 0.0001), percentagemotility (r= 0.38, P< 0.001), percentage of normal forms(r = 0.43, P < 0.001), sperm linear velocity (r= 0.42, P<0.001), and ATP concentration (r= 0.67, P< 0.0001). Folliclestimulating hormone (FSH) was found to be negatively correlatedwith the concentrations of both Tf (r= -0.31, P< 0.05) andof S-Tf-R (r= -0.45, P< 0.01). The mean concentration ofTf in seminal plasma was 50.4 µg/ml (35.9–67.2)in samples with normozoospermia (n= 22), and the concentrationwas significantly lower in patients with oligozoospermia (P<0.05), azoospermia of testicular origin (P < 0.001), andpost-vasectomy samples (P< 0.001). Seminal Tf was correlatedwith motile sperm concentration (r = 0.36, P< 0.001), percentageof motile spermatozoa (r= 0.25, P < 0.05), linear velocity(r= 0.24, P< 0.05) and ATP concentration (r= 0.44, P<0.001). The concentration of Tf was positively correlated withthat of S-Tf-R both in cases with spermatozoa present (r= 0.66,P < 0.001), and in cases with azoospermia of testicular origin(r = 0.51, P < 0.05) but not in vasectomy cases. It is concludedthat S-Tf-R in seminal plasma is a marker of spermatogenesisand may give information on the presence or absence of spermatogeneticcells in cases with azoospermia. Further investigations areneeded to assess its usefulness for clinical practice.     

Deterioration of sperm quality in young healthy Belgian men

We have retrospectively analysed the sperm characteristics of416 consecutive healthy young men who presented themselves inthe past 19 years as candidate sperm donors. Ejaculate volumeincreased slightly (P = 0.067), and average sperm concentrationdecreased (P = 0.035) by 12.4xlO6ml over the observation period,so that sperm count per ejaculate remained unchanged (P = 0.91).In contrast, sperm morphology (r = –0.23, P < 0.0001),rapid progressive motility (r = –0.42, P < 0.0001)and total motility (r = –0.33, P < 0.0001) presentedan important and time-related decrease. When a quadratic modelwas used rather than a linear one to analyse the data on rapidprogressive motility, there appeared to have been no furtherdecline since 1990. The average proportion of spermatozoa withnormal morphology decreased from 39.2% in the period 1977–1980to 26.6% in 1990–1995 (P < 0.0001), and the mean percentageof spermatozoa with rapid progressive motility decreased from52.7 to 31.7% (P < 0.0001). The percentage of candidate donorswith sperm characteristics below the 5th percentile cut-offvalue of a normal fertile population increased from 13 to 54%during the observation period (P < 0.0001). Since the techniqueof semen analysis has remained essentially unchanged in-so-faras has been practically possible, as has the method of recruitmentof candidate sperm donors, the observed deterioration of spermcharacteristics is considered to reflect degeneration of spermproduction among men aged between 20 and 40 years.