Human sperm volume regulation. Response to physiological changes in osmolality,channel blockers and potential sperm osmolytes

BACKGROUND: Volume regulation is an important sperm function because defective sperm cannot negotiate the female tract in an infertile mouse model and swollen human sperm cannot penetrate and migrate through mucus. METHODS AND RESULTS: The size of sperm from 52 donor ejaculates incubated in medium of female tract fluid osmolality (BWW290) was measured by flow cytometry to be identical to that in homologous semen osmolality (289-351 mosmol/kg), indicating effective volume regulation. Inhibition of anticipated regulatory volume decrease in BWW290 by the channel blocker quinine induced size increases and associated kinematic changes measured by computer-aided sperm analysis. Incubation in L-carnitine, myo-inositol and taurine did not change sperm volume or kinematics, but the presence of glutamate and K(+) decreased the efficiency of forward progression indicative of volume increase, suggesting them as potential osmolytes for human sperm. Linear regression suggested correlations of changes in cell volume and in kinematic parameters, and the association of faster forward progressive sperm with smaller cell size. CONCLUSIONS: Sperm volume and its regulation may be crucial to natural fertility. The identification of sperm osmolytes, ion channels and mechanisms involved would contribute to the understanding of male infertility and offer a lead for male contraception.     

Volume regulation of mature and immature spermatozoa in a primate model, and possible ion channels involved

BACKGROUND: Human ejaculated sperm undergo volume regulation, and swollen cells fail to penetrate mucus. Study of an infertile mouse model indicates maturation of volume regulation mechanism in the epididymis. METHODS: Sperm from the ejaculate and three regions of the epididymis of the cynomolgus monkey (Macaca fascicularis) were dispersed in BWW medium and changes in the cell volume and kinematics, and their responses to ion channel blockers, were monitored by flow cytometry and motion analysis. RESULTS: Initially swollen cauda epididymidal spermatozoa regained their original volume within 20 min, but not in the presence of 0.25 mM quinine. Corpus epididymidal spermatozoa underwent such regulatory volume decrease (RVD) to a lesser extent, with a similar response to quinine. Caput sperm showed no swelling throughout incubation. The chloride channel inhibitor NPPB also caused swelling of cauda spermatozoa and both quinine and NPPB decreased the efficiency of forward progression. RVD of ejaculated spermatozoa was inhibited by the K+ channel blockers quinine and 4-aminopyridine (4-AP) but not by tetraethylammonium, Ba2+ or Gd3+, or the specific potassium channel blockers charybdotoxin, margatoxin, dendrotoxin, apamin, glybenclamide or clofilium. Quinine and 4-AP also altered ejaculated sperm kinematics as reported in human ejaculated spermatozoa. CONCLUSIONS: Quinine- and 4-AP-sensitive (implying K+) and NPPB-sensitive (implying Cl-) channels are involved in RVD of primate sperm, which develop this volume regulatory ability in the epididymis.     

A role for the volume regulated anion channel in volume regulation in the murine CNS cell line,CAD

Aim: The role of the volume regulated anion channel (VRAC) in a model CNS neuronal cell line, CAD, was investigated. Methods: Changes in cell volume following hypotonic challenges were measured using a video-imaging technique. The effect of the Cl⁻ channel antagonists tamoxifen (10 μm ) and 4,4′-diisothiocyanatostilbene-2,2′-disulphonic acid (DIDS; 100 μm ) on regulatory volume decrease (RVD) were measured. The whole-cell voltage-clamp technique was used to characterize ICl_swell, the current underlying the VRAC. Results: Using the video-imaging technique, CAD cells were found to swell and subsequently exhibit RVD when subjected to a sustained hypotonic challenge from 300 mOsmol kg⁻¹ H₂O to 210 mOsmol kg⁻¹ H₂O. In the presence of tamoxifen (10 μm ) or DIDS (100 μm ) RVD was abolished, suggesting a role for the VRAC. A hypotonic solution (230 mOsmol kg⁻¹ H₂O) evoked ICl_swell, an outwardly rectifying current displaying time-independent activation, which reversed upon return to isotonic conditions. The reversal potential (E_rev) for ICl_swell was −14.7 ± 1.4 mV, similar to the theoretical E_rev for a selective Cl⁻ conductance. ICl_swell was inhibited in the presence of DIDS (100 μm ) and tamoxifen (10 μm ), the DIDS inhibition being voltage dependent. Conclusions: Osmotic swelling elicits an outwardly rectifying Cl⁻ conductance in CAD cells. The ICl_swell observed in these cells is similar to that observed in other cells, and is likely to provide a pathway for the loss of Cl⁻ which leads to water loss and RVD. As ischaemia, brain trauma, hypoxia and other brain pathologies can cause cell swelling, CAD cells represent a model cell line for the study of neuronal cell volume regulation.     

Characterization of potassium channels involved in volume regulation of human spermatozoa

Fertility depends in part on the ability of the spermatozoon to respond to osmotic challenges by regulating its volume, which may rely on the movement of K+. These experiments were designed to characterize the K+ channels possibly involved in volume regulation of human ejaculated spermatozoa by simultaneously exposing them to a physiological hypo-osmotic challenge and a wide range of K+ channel inhibitors. Regulation of cellular volume, as measured by flow cytometry, was inhibited when spermatozoa were exposed to quinine (QUI; 0.3 mM), 4-aminopyridine (4AP; 4 mM) and clofilium (CLO; 10 microM) which suggests the involvement of voltage-gated K+ channels Kv1.4, Kv1.5 and Kv1.7, acid-sensitive channel TASK2 and the beta-subunit minK (IsK) in regulatory volume decrease (RVD). QUI and 4AP and, to some extent, CLO also induced hyper activation-like motility. A sensitivity of RVD to pH could not be demonstrated in spermatozoa to support the involvement of TASK2 channels. Western blotting indicated the presence of Kv1.5, TASK2, TASK3 and minK channel proteins, but not Kv1.4. Furthermore, Kv1.5, minK and TASK2 were localized to various regions of the spermatozoa. Although Kv1.4, Kv1.7, TASK2 and TASK3 channels may have important roles in human spermatozoa, Kv1.5 and minK appear to be the most likely candidates for human sperm RVD, serving as targets for non-hormonal contraception.     

Andrology: Seminal volume and total sperm number trends in men attending subfertllity clinics in the Greater Athens area during the period 1977-1993

The trends for such Important parameters of male fertility asseminal volume and total sperm number were assessed in men livingpermanently in the Greater Athens area over a prolonged periodof time. To this end, the records of three andrological laboratoriesemploying the same method for semen evaluation were analysedretrospectively. Out of 23 850 men examined from 1977 to 1993(17 years) for couple subfertillty, a total of 2385 (10%) wereselected for evaluation by a randomization procedure. Analysisof the data included (i) estimation of mean seminal volume andtotal sperm number per year, (ii) assessment of percentage frequencydistribution for each seminal parameter and (iii) evaluationof seminal volume and total sperm number changes in relationto the year of observation and age of the subjects. A significantdecrease (P < 0.01) of total sperm number was observed overthe years with a mean (± SEM) of 154.3 ± 19.2x 10⁶ at the beginning (1977), dropping to 130.1 ± 13.3x 10⁶ in the final year (1993). Mean seminal volume was lowerin the final year of observation, but its difference from theinitial year value was not significant Frequency distributionanalysis showed a marked decline in the 240–400 x 10⁶sub-set of the range of sperm number values from 16.9 ±4.5% (1977) to 10.6 ± 1.6% in the final year (P <0.01). Multiple regression analysis of seminal volume, totalsperm number, age and year of assessment revealed a significantdecline of the two seminal parameters along the years of observation(P < 0.05 and P < 0.0001 respectively). Over the sameperiod, a marked deterioration of some air pollution indiceswas observed in that area. It is concluded that in this raciallyand ethnically homogeneous sample of men, living under the sameenvironmental conditions, a significant decline in seminal volumeand total sperm number occurred over the 17 years of observation.     

Role of estrogen and progesterone in the regulation of uterine peristalsis: results from perfused non-pregnant swine uteri

BACKGROUND: Adequate uterine contractility and peristalsis are involved in the transport of semen and gametes and in successful embryo implantation. Estrogen and progesterone fluctuate characteristically during the menstrual cycle. It has been suggested that both hormones influence uterine peristalsis in characteristic ways. METHODS: An extracorporeal perfusion model of the swine uterus was used that keeps the uterus in a functional condition and is suitable for the study of physiological questions. The effects of estrogen and progesterone on oxytocin-induced uterine peristalsis were assessed using an intrauterine double-chip microcatheter. RESULTS: Estrogen perfusion was associated with an increase in intrauterine pressure (IUP) in a dose-dependent manner. There was a significant difference between the IUP increase measured in the isthmus uteri and that in the corpus uteri, resulting in a cervico-fundal pressure gradient. Estrogen perfusion resulted in a significantly higher rate of peristaltic waves starting in the isthmus uteri and directed towards the corpus uteri. Progesterone was able to antagonize the estrogen effect in general. CONCLUSIONS: This study demonstrates that estrogen and progesterone have differential effects in the regulation of uterine peristalsis. The present observation shows that estrogen stimulates uterine peristalsis and is able to generate a cervico-fundal direction of peristalsis, whereas progesterone inhibits directed uterine peristalsis.     

ClC-3氯通道在二甲双胍抑制鼻咽癌细胞周期进程中的作用

目的:探讨ClC-3氯通道在二甲双胍抑制鼻咽癌细胞周期进程中的作用。方法:采用不同浓度二甲双胍处理低分化鼻咽癌细胞CNE-2Z,CCK-8法检测细胞活力,流式细胞术检测细胞周期分布,Western blot法检测ClC-3氯通道蛋白表达,全细胞膜片钳技术检测细胞氯电流。构建高表达ClC-3氯通道蛋白的质粒pEZ-M03-ClC-3转染CNE-2Z细胞,流式细胞术检测ClC-3氯通道对细胞周期分布的影响。结果:5、10和20 mmol/L浓度的二甲双胍均可有效抑制CNE-2Z细胞的活力。10 mmol/L二甲双胍可阻抑CNE-2Z细胞周期于G₀/G₁期,并抑制CNE-2Z细胞氯电流及ClC-3氯离子通道蛋白的表达。ClC-3氯通道蛋白高表达可逆转二甲双胍对CNE-2Z细胞周期分布的影响。结论:二甲双胍抑制鼻咽癌CNE-2Z细胞周期进程可能与抑制ClC-3氯通道功能和蛋白表达有关。     

IVF within microfluidic channels requires lower total numbers and lower concentrations of sperm

BACKGROUND: Microfluidic technology has been utilized in numerous biological applications specifically for miniaturization and simplification of laboratory techniques. We sought to apply microfluidic technology to murine IVF. METHODS: Microfluidic devices measuring 500 microm wide, 180 microm deep, and 2.25 cm in length were designed and fabricated using poly(dimethylsiloxane) (PDMS). Controls were standard centre-well culture dishes with 500 microl of media, half of which also contained PDMS as a material control. Denuded mouse oocytes were placed into microchannels or centre-well dish controls in groups of 10, then co-incubated overnight with epididymal mouse sperm at various concentrations. Fertilization was assessed and Fisher's exact test was used for statistical analysis (P < 0.05 significant). RESULTS: Fertilization rates between the two control groups (42%, no PDMS; 41%, with PDMS; not significant) were similar. Fertilization rates for denuded oocytes at standard mouse insemination sperm concentration (1 degrees 10(6) sperm/ml) was poorer in microchannels (12%) than controls (43%; P < 0.001). As insemination concentrations decreased, fertilization rates improved in microchannels with a plateau between 8 degrees 10(4) and 2 degrees 10(4) sperm/ml (4000-1000 total sperm). At these concentrations, combined fertilization rate for denuded oocytes was significantly higher in microchannels than centre-well dishes (27 versus 10%, respectively; P < 0.001), and was not significantly different from corresponding controls with a sperm concentration of 1 degrees 10(6) (37%; P = 0.06). CONCLUSIONS: Murine IVF can be conducted successfully within microfluidic channels. Lower total numbers and concentrations of sperm are required. Microfluidic devices may ultimately be useful in clinical IVF.     

A role for tachykinins in the regulation of human sperm motility

BACKGROUND: Tachykinins and tachykinin receptors are widely distributed in the male reproductive tract and appear to be involved in reproduction. However, the function and expression of tachykinins and their receptors in human spermatozoa remain poorly studied. We analysed the effects of tachykinins on sperm motility and characterized the population of tachykinin receptors in human spermatozoa. METHODS AND RESULTS: Motility analysis was performed following World Health Organization guidelines and we found that substance P (SP), human hemokinin-1 (hHK-1), neurokinin A (NKA) and neurokinin B (NKB) produced concentration-dependent increases in sperm progressive motility. The effects of tachykinins were antagonized by the NK(1) receptor-selective antagonist SR 140333, the NK(2) receptor-selective antagonist, SR 48968 and, to a lesser extent, also by the NK(3) receptor-selective antagonist SR 142801. Immunocytochemistry studies showed expression of the NK(1), NK(2) and NK(3) tachykinin receptor proteins in spermatozoa with different major sites of localization for each receptor. Western blot analysis confirmed the presence of tachykinin receptors in sperm cell homogenates. RT-PCR demonstrated expression of the genes that encode SP/NKA (TAC1), NKB (TAC3) and hHK-1 (TAC4) but not the genes TACR1, TACR2 and TACR3 encoding NK(1), NK(2) and NK(3) receptors, respectively. CONCLUSIONS: These results show for the first time that the NK(1), NK(2) and NK(3) tachykinin receptor proteins are present in human spermatozoa. Our findings suggest that tachykinins, probably acting through these three tachykinin receptors, play a role in the regulation of human sperm motility.     

Uterine hyperperistalsis and dysperistalsis as dysfunctions of the mechanism of rapid sperm transport in patients with endometriosis and infertility

Women suffering from infertility in association with mostlymild endometriosis were subjected to vaginal sonography of uterineperistalsis during the menstrual period, the early, mid- andlate follicular phases, and the mid-luteal phase of the menstrualcycle. The data obtained were compared with those of healthycontrols. Women with endometriosis displayed a marked uterinehyperperistalsis that differed significantly from the peristalsisof the controls during the early and mid-follicular and mid-lutealphases. During the late follicular phase of the cycle, uterineperistalsis in women with endometriosis became dysperistaltic,arrhythmic and convulsive in character, while in controls peristalsiscontinued to show long and regular cervico-fundal contractions.Hysterosalpingoscintigraphy during the early, mid- and latefollicular phases revealed that hyperperistalsis in the earlyand mid-follicular phases of patients with endometriosis resultedin a dramatic increase in the transport of inert particles fromthe vaginal depot, through the uterus into the tubes and alsointo the peritoneal cavity. During the late follicular phaseof the cycle, the dysperistalsis observed in women with endometriosisresulted in a dramatic reduction of uterine transport capacityin comparison with the healthy controls. We consider uterinehyperperistalsis to be the mechanical cause of endometriosisrather than retrograde menstruation. Dysperistalsis in the latefollicular phase of patients with endometriosis may compromiserapid sperm transport Uterine hyperperistalsis and dysperistalsisare considered to be responsible for both reduced fertilityand the development of endometriosis.     

Effect of hyperosmolarity and furosemide on resting membrane potential and volume of rat skeletal muscle fibers

S. V. Kurashov, Kazan' Medical Institute. Presented by Academician of the Academy of Medical Sciences of the USSR A. D. Ado.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 108, No. 11, pp. 563–566, November, 1989.     

Study of mitochondrial membrane potential, reactive oxygen species, DNA fragmentation and cell viability by flow cytometry in human sperm

BACKGROUND: Sperm cell death appears to be a cause of male infertility. The objective of this study was to determine the most reliable method for the evaluation of sperm quality in semen samples during sperm preparation for IVF. METHODS: Conventional analysis of semen samples was compared with several cytofluorometric methods detecting death-associated changes. Neat semen from infertile patients and sperm prepared by PureSperm gradient were studied by conventional microscopy and analysed for mitochondrial membrane potential (Delta Psi(m)), generation of reactive oxygen species, DNA fragmentation and cell viability. RESULTS: In neat semen, a positive correlation was found between the percentage of Delta Psi(m)(high) sperm cells and standard semen parameters (concentration/motility). Sperm cells depicting Delta Psi(m)(high) and cells with low DNA fragmentation displayed high fertilization rate after IVF. The only changes that could be detected in prepared sperm were changes in Delta Psi(m), with Delta Psi(m)(high) sperm positively correlated with forward motility and also with high fertilization rates after IVF. CONCLUSION: Analysis of mitochondrial membrane potential is the most sensitive test by which to determine sperm quality. These findings promise development of a test that may help to predict successful IVF.     

Automated semen analysis shows an increase in sperm concentration and motility with time in Makler chambers having excess sample volume

The sperm concentration and motility within a Makler chamber increased markedly after 3-5 min of settling when one large drop (approximately 24 microliters) of semen was loaded in the chamber. The values remained stable when a 5-microliter volume was loaded and were intermediate with a 10-microliter volume. In order to obtain accurate data, it is necessary to load the Makler chamber with a semen volume of no more than 5 microliters.     

The presence of heparan sulfate in the mammalian oocyte provides a clue to human sperm nuclear decondensation in vivo

BACKGROUND: Previous results from our laboratory have led us to proposeheparan sulfate (HS) as a putative protamine acceptor duringhuman sperm decondensation in vivo. The aim of this paper wasto investigate the presence of glycosaminoglycans in the mammalianoocyte in an effort to better support this contention. METHODS: Two experimental approaches are used: oocyte labeling to identifythe presence of HS and analysis of sperm decondensing abilityof fresh oocytes in the presence or absence of specific glycosidases. RESULTS: Staining of mouse zona-intact oocytes with the fluorescent cationicdye, Rubipy, at pH 1.5 allowed for the detection of sulfateresidues in the ooplasm by confocal microscopy. HS was detectedin the ooplasm by immunocytochemistry. A sperm decondensationmicroassay using heparin and glutathione was successfully developed.The same level of sperm decondensation could be attained whenheparin was replaced by mouse zona-free oocytes. Addition ofheparinase to the oocyte/glutathione mixture significantly reducedsperm decondensation (P = 0.0159), while there was no effectfollowing addition of either chondroitinase ABC or hyaluronidase. CONCLUSIONS: The results presented in this paper demonstrate for the firsttime that HS is present in the mammalian oocyte and show thatHS is necessary for fresh oocytes to express their sperm decondensingability in vitro.     

Enhancement of mouse sperm motility by the PI3-kinase inhibitor LY294002 does not result in toxic effects on preimplantation embryo development

BACKGROUND: A reduced number of progressively motile sperm (as may occur in cases of asthenozoospermia or when cryopreserved spermatozoa are used for fertilization) limits the possibility of applying various assisted reproductive techniques (ARTs). We previously showed that incubation of sperm with the phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 increases sperm progressive motility and enhances the number of sperm recovered by capacitation protocols used in ART. METHODS AND RESULTS: In the present study, we investigate the motility-enhancing effects of this compound in epididymal mouse sperm, and examine the use of the mouse system to investigate the effect of LY294002 on oocyte fertilization and preimplantation embryo development. Our results show that neither pre-incubation of mouse spermatozoa with the inhibitor during in vitro capacitation nor the direct addition of LY294002 to the sperm-oocyte mixture significantly affects the process of fertilization and preimplantation development of embryos produced even when they developed in the presence of LY294002. CONCLUSIONS: The present data encourage the design of new drugs based on the molecular structure of LY294002, which may open up new options for the in vitro treatment of human/animal asthenozoospermia.     

Sonographic evidence for the involvement of the utero-ovarian counter-current system in the ovarian control of directed uterine sperm transport

Sperm transport from the cervix into the tube is an important uterine function within the process of reproduction. This function is exerted by uterine peristalsis and is controlled by the dominant ovarian structure via a cascade of endocrine events. The uterine peristaltic activity involves only the stratum subvasculare of the myometrium, which exhibits a predominantly circular arrangement of muscular fibres that separate at the fundal level into the fibres of the cornua and continue into the circular muscles of the respective tubes. Since spermatozoa are transported preferentially into the tube ipsilateral to the dominant follicle, this asymmetric uterine function may be controlled by the ovary via direct effects utilizing the utero-ovarian counter-current system, in addition to the systemic circulation. To test this possibility the sonographic characteristics of the uterine vascular bed were studied during different phases of the menstrual cycle. Vaginal sonography with the measurement of Doppler flow characteristics of both uterine arteries and of the arterial anastomoses of the uterine and ovarian arteries (junctional vessels) in the cornual region of both sides of the uterus during the menstrual phase of regular-cycling women demonstrated significant lower resistance indices of the junctional vessels ipsilateral to the side of the dominant ovarian structure as compared with the corresponding arteries contralaterally. By the use of the perfusion mode technique, it could be observed that vascular perfusion of the fundal myometrium was significantly increased ipsilateral to the dominant follicle during the late follicular phase of the cycle. These results show that the endocrine control of the dominant ovarian structure over uterine function is not only exerted via the systemic circulation but also directly, most probably utilizing the utero-ovarian counter-current system.     

Detection of apoptotic alterations in sperm in subfertile patients and their correlations with sperm quality

BACKGROUND: The aim of the present study was to define the effect of apoptosis on sperm quality and function. METHODS: The apoptotic features in sperm were assessed in 60 subfertile subjects, using Annexin-V staining for phosphatidylserine (PS) externalization and Tdt-mediated dUTP nick end labelling (TUNEL) assay for DNA fragmentation. RESULTS: On average, about 45% of the sperm were found to be apoptotic based on the results from Annexin-V staining, including both early (Annexin-V-positive, PI-negative) and late apoptosis (Annexin-V-positive, PI-positive). TUNEL-positive cells (median value 15%) significantly correlated to late apoptosis but not early apoptosis, indicating that DNA fragmentation only occurs at the later stage of sperm apoptosis. TUNEL-positive and late apoptotic cells (Annexin-V-positive, PI-positive) were found to be inversely correlated to sperm motility and vitality, and positively to abnormal sperm morphology. On the other hand, it is surprising to note that the apoptotic alterations in sperm positively correlated to sperm concentration or total sperm counts. CONCLUSIONS: Overall results from this study support the abortive apoptosis theory; apoptosis in mature sperm is initiated during spermatogenesis, after which some cells earmarked for elimination via apoptosis may escape the removal mechanism and contribute to poor sperm quality.     

The role of sperm proteasomes during sperm aster formation and early zygote development: implications for fertilization failure in humans

BACKGROUND Sperm aster organization during bovine and human fertilization requires a paternally-derived centriole that must first disengage from the sperm tail connecting-piece. We investigated the participation of the 26S proteasome in this process. METHODS Proteasome localization and enzymatic activity were studied in normal and pathological human spermatozoa by immunocytochemistry and enzyme-substrate assays. The role of proteasomes during bovine zygote development was investigated using a pharmacological proteasome-inhibitor, MG132, and with anti-proteasome antibodies delivered by Streptolysin O-permeabilization or with the Chariot reagent. Human zygotes discarded after ICSI failures (n = 28) were also examined. RESULTS Proteasomes were localized in the sperm acrosome and connecting-piece, as well as in the pronuclei of bovine and human zygotes. Proteasomal enzymatic activities were decreased in defective human spermatozoa. Disrupted sperm aster formation and pronuclear development were found after pharmacological and immunological block of proteasomes in human/bovine spermatozoa and oocytes, as well as in 28 discarded human post-ICSI fertilization failures. CONCLUSIONS Specific proteasome inhibition disrupts sperm aster formation and pronuclear development/apposition in bovine and human zygotes. Human spermatozoa with defective centriolar/pericentriolar structures have decreased proteasomal enzymatic activity. Release of a functional sperm centriole that acts as a zygote microtubule-organizing center probably relies on selective proteasomal proteolysis. These findings suggest an important role of sperm proteasomes in zygotic development.     

Role of sperm FISH studies in the genetic reproductive advice of structural reorganization carriers

The use of fluorescence in-situ hybridization (FISH) in decondensed spermatozoa from carriers of structural chromosomal abnormalities provides a way to estimate the amount of unbalanced products. This methodology has become a tool of special interest for a better approximation of the reproductive competence of the carriers. Although there is no discussion regarding the cytogenetic value of the information obtained, the usefulness of performing individual sperm FISH studies must be weighed depending on the object of the study. In this paper, we introduce some considerations concerning the convenience of a routine application of sperm FISH analysis in the major populations of structural reorganization carriers. For each group, the significance of the information that can be obtained and its relevance for genetic reproductive advice are discussed.     

Chromosome analysis of epididymal and testicular sperm in azoospermic patients undergoing ICSI

BACKGROUND: Although ICSI provides a way of treating azoospermic men, concern has been raised about the potential risk for transmission of genetic abnormalities to the offspring. We quantified the incidence of chromosomal abnormalities in epididymal and testicular sperm retrieved from azoospermic patients undergoing ICSI. METHODS: Individual testicular sperm were collected from testicular biopsies with an ICSI pipette, and epididymal sperm were retrieved by microsurgical epididymal sperm aspiration. Samples were processed by fluorescent in-situ hybridization (FISH) for chromosomes 18, 21, X and Y and the results compared with those from normal ejaculated samples. RESULTS: The overall aneuploidy rate of 11.4% in men with non-obstructive azoospermia was significantly higher (P = 0.0001) than the 1.8% detected in epididymal sperm from men with obstructive azoospermia and also the 1.5% found in ejaculated sperm. No significant difference was found between the epididymal and ejaculated samples. When the chromosomal abnormalities were analysed, gonosomal disomy was the most recurrent abnormality in both obstructive and non-obstructive azoospermic patients, while autosomal disomy was the most frequent in ejaculated sperm. CONCLUSIONS: Sperm of non-obstructive azoospermic men had a higher incidence of chromosomal abnormalities, of which sex chromosome aneuploidy was the most predominant. Genetic counselling should be offered to all couples considering infertility treatment by ICSI with testicular sperm.