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.     

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.     

SHORT COMMUNICATION: Origin, development and ultrastructure of boar spermatozoa with folded tails and with two tails

Speramtozoa from the three epididymal regions (head, body andtail) of healthy and sexually mature boars been examined bylight miroscopy, and scanning and transmission elctron and internalmorphologies of aberrant spermatozoa with folded tails and spermatozoawith one or two heads and two fused tails and spermatozoa withone or two heads and two fused tails have been established.A count carried out in each region of the epididymis indicatedthat significant differences(P >0.01) exist in the frequenciesof each type of malformation and the epididymal region fromwhich the spermatozoa in the cauda of the epididymis from immaturespermatozoa that have not ejected the distal cytoplasmic droplet.The plasma membrane which covers the main piece is fused withthe membranes of the midpiece, the connecting piece and thehead. The fibrous sheath deforms the connectiong piece and thehead. the fibrous sheath deforms the mitochondrial sheath andisplaced between the plasma membrane and the postacrosomal denselamina. Spermatozoa with one head and two fused tails originatein the epididyaml body from spermatozoa with one head and twounfused tails coming from the cephalic region of the edididymis.Spermatozoa with two heads and two fused tails originate inthe cephalic region of the epididymis by head-to-head agglutinationof two spermatozoa with two fused tails increases as they progressthrough the epidididymal duct. Their tails, parallel in monocephalicspermatozoa and helicoid in bicephalic spermatozoa, have twocomplete axonemal axes. In their midpiece, the mitochondrialsheaths of the two axes are fused, producing an 8-shapedsheath.     

Water transport between CNS compartments: functional and molecular interactions between aquaporins and ion channels

The physiological ability of the mammalian CNS to integrate peripheral stimuli and to convey information to the body is tightly regulated by its capacity to preserve the ion composition and volume of the perineuronal milieu. It is well known that astroglial syncytium plays a crucial role in such process by controlling the homeostasis of ions and water through the selective transmembrane movement of inorganic and organic molecules and the equilibration of osmotic gradients. Astrocytes, in fact, by contacting neurons and cells lining the fluid-filled compartments, are in a strategic position to fulfill this role. They are endowed with ion and water channel proteins that are localized in specific plasma membrane domains facing diverse liquid spaces. Recent data in rodents have demonstrated that the precise dynamics of the astroglia-mediated homeostatic regulation of the CNS is dependent on the interactions between water channels and ion channels, and their anchoring with proteins that allow the formation of macromolecular complexes in specific cellular domains. Interplay can occur with or without direct molecular interactions suggesting the existence of different regulatory mechanisms. The importance of molecular and functional interactions is pinpointed by the numerous observations that as consequence of pathological insults leading to the derangement of ion and volume homeostasis the cell surface expression and/or polarized localization of these proteins is perturbed. Here, we critically discuss the experimental evidence concerning: (1) molecular and functional interplay of aquaporin 4, the major aquaporin protein in astroglial cells, with potassium and gap-junctional channels that are involved in extracellular potassium buffering. (2) the interactions of aquaporin 4 with chloride and calcium channels regulating cell volume homeostasis. The relevance of the crosstalk between water channels and ion channels in the pathogenesis of astroglia-related acute and chronic diseases of the CNS is also briefly discussed.     

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.     

Maturation of human spermatozoa (from selected epididymides of prostatic carcinoma patients) with respect to their morphology and ability to undergo the acrosome reaction

Spermatozoa were recovered form three regions of the epididymisof six prostatic carcinoma patients. After washing and incubatingfor 3 h in Ham's F-10 medium, with or without 5 µM A23187for the last 30 min, spermatozoa were tested for vitality byhypotonic swelling and permeated with methanol to detect theacrosome with peanut agglutinin. Whereas the extent of spontaneousacrosome reactions was similar for spermatozoa from all regionsof the duct, 17 and 28% of spermatozoa from all regions of theduct, 17 and 28% of spermatozoa from the corpus and cauda epididymidisrespectively, responded to stimulation by A23187 with acrosomereactions but there was no stimulation by A23187 of spermatozoafrom the efferent ducts. The percentage of morphologically normalspermatozoa increased stepwise towards the distal regions, withabnormalities being mostly enlarged heads in more proximal regions:they were largely absent form the cauda epididymidis. Spermhead swelling was similarly observed in cynomolgus monkey spermatozoafrom the caput epididymidis but not the more distal regions.These forms were not observed when spermatozoa were fixed beforesmearing, indicating that they were artefacts of sperm preparation.The changes in the susceptibility of non-fixed epididymal spermatozoato produce morphological artefacts and the gain in their acrosomalresponse to ionophore demonstrate maturational changes of spermatozoain the human epididymis.     

HE1/NPC2 status in human reproductive tract and ejaculated spermatozoa: consequence of vasectomy

We have previously demonstrated that the amount of HE1/NPC2 mRNA and protein expressed in the human epididymis is decreased under vasectomy. In this study, western blot analyses showed that many vasovasostomized men are characterized by high HE1/NPC2 levels in spermatozoa when compared with fertile donors. HE1/NPC2 association with sperm from vasovasostomized men was not related to low motility per se as spermatozoa from asthenospermic men have HE1/NPC2 levels similar to those in normal fertile semen samples. Spermatozoa from vasovasostomized men with high amount of HE1/NPC2 are characterized by higher concentration of cholesterol and more lipid raft domains. HE1/NPC2 is secreted in different glycoforms by different tissues of human male reproductive tract. These forms are due to variation in N-glycosylation, and only the deglycosylated form is associated with spermatozoa from some vasovasostomized men. Compared with normal men, seminal plasma of vasectomized men is characterized by a major decrease in immunodetectable HE1/NPC2 without change in the glycosylation pattern. Following surgical vasectomy reversal, seminal plasma HE1/NPC2 was found in similar amounts to the ones characterizing normal men. Considering the potential role of HE1/NPC2 in cholesterol transport during sperm maturation, unusual high levels of this protein associated with spermatozoa of vasovasostomized men may reflect epididymal sequelae occurring when the vas deferens is obstructed.     

The relative viability of human spermatozoa from the vas deferens, epididymis and testis before and after cryopreservation

Testicular and epididymal spermatozoa are routinely used with in-vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) to achieve pregnancies. In addition, excess cryopreserved spermatozoa can be thawed and used for ICSI. However, information on the recovery of epididymal and testicular spermatozoa after freeze-thaw is lacking. This is important to determine the feasibility of using previously cryopreserved aspirated spermatozoa for ICSI. We prospectively compared the viability of fresh and frozen-thawed spermatozoa from the vas deferens, epididymis and testicle by several measures. Testis spermatozoa were obtained from men with non-obstructive azoospermia (n = 5), epididymal spermatozoa from men with obstructive azoospermia (n = 8), and vasal spermatozoa from fertile men by vasal irrigation at vasectomy (n = 5). The viability of fresh spermatozoa was assessed by motility, two vital stains (carboxyfluorescein, 0.08 mg/ml and propidium iodide, 20 mg/ml) and the hypo-osmotic swelling assay (HOS; 100 mmol/l citrate and fructose). After cryopreservation, spermatozoa were thawed and all viability measures repeated. Although fresh vasal spermatozoa were the most motile, testicular spermatozoa exhibited similar, high viability (91 and 86% respectively) by vital stain. Spermatozoa from testis, epididymis and vas deferens survived cryopreservation equally well by vital stain, but not by motility. As a selection measure, the HOS assay identified significantly more viable epididymal and testicular spermatozoa than did motility in both fresh and frozen-thawed populations. It appears feasible to use frozen-thawed extracted spermatozoa for ICSI when motility and a selection measure such as the HOS assay are used. With fresh testis spermatozoa, selection methods may not be necessary prior to ICSI, as cell viability is high.     

Role of volume-stimulated osmolyte and anion channels in volume regulation by mammalian sperm

The ability to maintain cellular volume is an important general physiological function. Swelling induced by hypotonic stress results in the opening of channels, through which ions exit with accompanying water loss (regulatory volume decrease, RVD). RVD has been shown to occur in mammalian sperm, primarily through the opening of quinine-sensitive potassium channels. However, as yet, direct evidence for the participation of anion channels in sperm RVD has been lacking. The chloride channel type ClC-3 is believed to be involved in RVD in other cell types. Using electronic cell sizing for cell volume measurement, the following results were obtained. (i) The anion channel blockers 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB), tamoxifen and 4,4'-diisothiocyanostilbene-2,2'-disulphonic acid (DIDS) increased hypotonic swelling in concentration-dependent fashion, whereas verapamil (P-glycoprotein inhibitor) had little effect. The most potent, NPPB and DIDS, blocked RVD without affecting cell membrane integrity at effective concentrations. (ii) When gramicidin was included to dissipate Na+/K+ gradients, major secondary swelling was observed under hypotonic conditions. This secondary swelling could be reduced by NPPB, and suppressed completely by replacing chloride in the medium with sulphate, an ion which does not pass through chloride channels. It was deduced that the initial hypotonic swelling activated an anion channel through which chloride ions could then enter freely down a concentration gradient, owing to the lack of a counter-gradient of potassium. (iii) Taurine, an osmolyte often involved in RVD, does not appear to play a role in sperm RVD because lengthy preincubation with taurine did not alter sperm RVD response. Our observations provide direct evidence that a chloride channel (possibly ClC-3) is involved in the process of volume regulation in mammalian sperm.     

Role of potassium and chlorine channels in the regulation of thymocyte volume in rats

Regulatory decrease in thymocyte volume under conditions of osmotic stress was abolished by potassium and chlorine channel blockers. Osmotic stress-activated chlorine channels belong to 2 pharmacological types. The maxi-anion channel is sensitive to Gd³⁺. The volume-sensitive outwardly rectifying chlorine channel is inhibited with glybenclamide and phloretin. __________ Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 145, No. 5, pp. 544–547, May, 2008     

Calcium signalling in human spermatozoa: a specialized 'toolkit' of channels, transporters and stores

Ca(2+) is a ubiquitous intracellular messenger which encodes information by temporal and spatial patterns of concentration. In spermatozoa, several key functions, including acrosome reaction and motility, are regulated by cytoplasmic Ca(2+) concentration. Despite the very small size and apparent structural simplicity of spermatozoa, evidence is accumulating that they possess sophisticated mechanisms for regulation of cytoplasmic Ca(2+) concentration and generation of complex Ca(2+) signals. In this review, we consider the various components of the Ca(2+)-signalling 'toolkit' that have been characterized in somatic cells and summarize the evidence for their presence and activity in spermatozoa. In particular, data accumulated over the last few years show that spermatozoa possess one (and probably two) Ca(2+) stores as well as a range of plasma membrane pumps and channels. Selective regulation of the various components of the 'toolkit' by agonists probably allows spermatozoa to generate localized Ca(2+) signals despite their very small cytoplasmic volume, permitting the discrete and selective activation of cell functions.     

Delivery following intracytoplasmic injection of mature sperm cells recovered by testicular fine needle aspiration in a case of hypergonadotropic azoospermia due to maturation arrest

This is the first reported delivery following intracytoplasmicsperm injection (ICSI) of mature live testicular sperm cellscollected in a case of hypergonadotrophic azoospermia with maturationarrest. The 30 year old couple presented with primary infertilityof 11 years duration, the man being submitted in childhood tofive orchidopexy operations for the treatment of cryptorchism.He had elevated serum follicle stimulating hormone (FSH; 18.8IU/1), an atrophic left testis and a normal sized right testis,the biopsy of which diagnosed maturation arrest and focal scarring.The couple refused donor insemination for religious reasonsand the only option was an attempt at testicular sperm collection.Multiple testicular and epididymal fine needle aspirations wereperformed, using an aspiration handle loaded with 20 ml syringeand 21–23 gauge butterfly needles. The mature spermatozoarecovered were used to inseminate the oocytes by ICSL Priorto this procedure, the patient‘s wife underwent ovulationinduction using a long protocol of mid-Iuteal gonadotrophin-releasinghormone analogue/human menopausal gonadotrophin (GnRHa/HMG).At oocyte retrieval, ten oocytes were recovered. Eight livesperm cells were recovered from the aspirates of the right testis.Following ICSI into four metaphase II and two metaphase I oocytes,one mature oocyte was fertilized, cleaved and was transferredto the uterus 48 h after oocyte retrieval. The patient conceivedand delivered a 3300 g boy at term. In conclusion, our resultsdemonstrate that this novel approach should be considered incases with hypergonadotrophic azoospermia due to testicularfailure. Further experience is needed to establish the exactcriteria for its use.     

Comparative study of the effect of human cervical mucus and a cervical mucus substitute, Healonid, on capacitation and the acrosome reaction of human spermatozoa in vitro

The present study was designed to investigate the effect ofhuman cervical mucus on capacitation and the acrosome reactionof human spermatozoa and compare its effect to that of a cervicalmucus substitute, sodium hyaluronate (Healonid). Spermatozoafrom donors of proven fertility were isolated from semen usingcervical mucus, Healonid or a direct swim-up (acting as thecontrol). Sperm capacitation and the acrosome reaction weremonitored by the chlortetracycline assay. In the mucus-treatedgroup, there was a significantly higher percentage of capacitatedspermatozoa, but a low incidence of spontaneous and A23187-inducedacrosome reactions compared to the control. The use of Healonidduring sperm isolation mimicked the effect of mucus relativelysuccessfully. Since mucus and Healonid show very little chemicalsimilarity, this finding would imply that cervical mucus exertsa physical effect during its interaction with spermatozoa, althougha chemical effect cannot be completely dismissed. In conclusion,this study confirms early reports describing the ability ofcervical mucus to capacitate spermatozoa but at the same timeconserve sperm function. The finding that Healonid exerts analmost identical effect on spermatozoa would lend support toits use as a cervical mucus substitute during in-vitro fertilityassessments and research studies.     

酸敏感离子通道基因重组质粒的构建、表达及其活性测定

为了构建含有小鼠酸敏感离子通道(ASIC1a、ASIC2a)基因全长cDNA的重组质粒,并表达有生物学活性的ASIC1a和ASIC2a融合蛋白,本研究通过逆转录聚合酶链反应(RT-PCR)分别获得小鼠ASIC1a和ASIC2a全长cDNA,并将其克隆入真核表达载体pEGFP-N3中,构建含小鼠全长ASIC1a和ASIC2a基因的重组质粒pEGFP-ASIC1a和pEGFP-ASIC2a,经DNA测序鉴定序列正确。脂质体法分别将其转染至中国仓鼠卵巢细胞(CHO),荧光显微镜下观察小鼠ASIC1a和ASIC2a融合蛋白在细胞内的表达分布,Westernblot检测其蛋白表达。酸性处理转染重组质粒细胞,并比较其细胞存活率及乳酸脱氢酶(LDH)的释放来评价融合蛋白的生物学活性。结果显示:本研究成功地分别将小鼠ASIC1a和ASIC2a全长cDNA克隆入pEGFP-N3载体中,并将其转染至CHO细胞,荧光显微镜下观察到CHO细胞膜周围呈强绿色荧光条带,Westernblot显示小鼠ASIC1a和ASIC2a融合蛋白分别在90kD和88kD处有表达。经酸性处理的转染ASIC1a和ASIC2a重组质粒的CHO细胞,分别较其在pH7.4条件下的细胞存活率明显降低,LDH释放量明显增高,且通道阻断剂可抑制该效应。上述结果提示,我们已成功构建出含小鼠ASIC1a和ASIC2a全长cDNA的重组质粒pEGFP-ASIC1a和pEGFP-ASIC2a,并使有生物学活性的ASIC1a和ASIC2a融合蛋白在CHO细胞中表达。     

Andrology: Intracytoplasmic injection of human spermatozoa into mouse oocytes: a useful model to investigate the oocyte-activating capacity and the karyotype of human spermatozoa

When intracytoplasmic sperm injection (ICSI) is performed, itis important to know the capacity of sperm cells to activatethe oocytes, although knowledge of their ability to fuse withthe oocytes is not vital. Hamster oocytes are not suitable forthis purpose because they are easily activated by the injectionprocedure itself. We therefore investigated whether mouse oocytescould be used to assess the activation properties of human spermatozoa.Mouse oocytes were randomized for injection with initially motilespermatozoa, medium, heat-treated or salt-extracted spermmatozoa,and the survival and activation rates were examined. About halfof the mouse oocytes survived the intracytoplasmic injectionof a human sperm cell. Unlike hamster oocytes, the rate of activationprovoked by the injection procedure itself was acceptably low(20%), resembling in this respect the behaviour of human oocytes.Following the injection of initially motile human spermatozoa,all mouse oocytes were activated. The injection of heat-treatedor salt-extracted human spermatozoa resulted in activation ratesof 14 and 15% respectively, comparable with the results followingsham ICSI. These data support the hypothesis of a sperm-associatedoocyte activation factor. In most activated oocytes, the humansperm nucleus decondensed to form a male pronucleus. Cytogeneticanalysis at the first metaphase revealed that human sperm chromosomeswere able to undergo replication in a heterologous environment.From our data we concluded that human spermatozoa can be injectedsuccessfully into mouse oocytes, resulting in a reasonable survivalrate, and that mouse oocytes provide a useful model for boththe assessment of the sperm-associated oocyte activation factorand the cytogenetic analysis of human spermatozoa.     

G-CSF对豚鼠单个缺血心房肌细胞钙、钠、钾电流的影响

目的:采用膜片钳全细胞记录方法,观察粒细胞集落刺激因子(G-CSF)急性干预对分离的豚鼠单个缺血心房肌细胞钙、钠、钾电流的影响。方法:使用酶解方法获得豚鼠单个心房肌细胞,在缺血缺氧条件下,采用全细胞膜片钳技术观察记录不同浓度G-CSF急性干预对钙通道电流-电压曲线、激活曲线、失活曲线,钠通道电流-电压曲线、激活曲线、失活曲线、静态失活曲线,延迟整流钾通道及其快成分、慢成分电流-电压曲线的影响。结果:随G-CSF浓度的增加,缺血心房肌细胞膜L型钙通道电流较缺血对照组有明显增大。当G-CSF浓度超过100μg/kg后,L型钙通道电流的增强维持于同一水平。当给予G-CSF浓度为300μg/kg时最大激活电压发生改变,较前有所增加。激活曲线与失活曲线未见明显改变。不同浓度G-CSF对缺血心房肌细胞膜钠离子通道电流无明显影响。G-CSF干预缺血心房肌细胞膜延迟整流钾电流未见明显改变,但延迟整流钾电流快成分在G-CSF 100μg/kg干预后明显增加,而延迟整流钾电流慢成分未见明显改变。结论:G-CSF对于缺血心房肌细胞部分通道的作用影响基本为非电压依赖性,但具有浓度依赖性,可能减少缺血心房心律失常的发生率。     

'9 + 0' axoneme in spermatozoa and some nasal cilia of a patient with totally immotile spermatozoa associated with thickened sheath and short midpiece

A patient is described with 100% immotile spermatozoa. The sperm cells lack the central pair of microtubules or the complete axoneme. This defect is associated with a thickened fibrous sheath and a shortened midpiece containing defective mitochondria. Fifteen per cent of the nasal cilia of this patient lack the central pair of microtubules. Eighteen similar cases have been described in the literature suggesting that this association of ultrastructural defects may be considered a syndrome.     

Ovary and ovulation: In-vitro maturation, fertilization and embryo development of immature oocytes from early preantral follicles from prepuberal mice in a simplified culture system

A simplified culture system was developed for the in-vitro maturationof early preantral mouse ovarian follicles. The follicles werecultured singly in 20 (µ droplets under oil in mediumsupplemented with recombinant follicle stimulating hormone (r-FSH)at 37°C and 5% CO² in air. The follicles grew and becameattached to the bottom of the dish, progressively lost theirspherical structure by outgrowth of the granulosa cells throughthe basal membrane and developed follicles with antral-likecavities. The normal three-dimensional follicular structurewas lost but all components, i.e. theca, granulosa and oocyte,remained functional, as was proven by the oestradiol, inhibinand progesterone secretion patterns. Follicle survival exceeded80% and histological analysis proved the absence of atresiaand cell death in granulosa cells up to day 16. Oocytes of 55(± 4) µm diameter on the day of isolation reached74 (± 3) µm by day 16 of culture. The optimal momentfor inducing the final meiotic maturation with human chorionicgonadotrophin was investigated: the highest absolute numbersof metaphase II oocytes were obtained on days 12 and 14 (39and 41%). The fertilizing potential of the in-vitro maturedoocytes was comparable to in-vivo matured controls. A 50% hatched-blastocystdevelopment rate was observed.     

Andrology: Epididymal sperm aspiration and use of donor spermatozoa when necessary for in-vitro fertilization, and vasoepididymostomy: an acceptable combination in the treatment of obstructive azoospermia

Infertility due to obstructive azoospermia in 24 men was treatedwith a combination of scrotal exploration, microsurgical spermaspiration and vasoepididymostomy, at the same operation. In-vitrofertilization (IVF) and embryo transfer were performed usingepididymal spermatozoa. Donor spermatozoa were used if no motileepididymal spermatozoa were obtained. With this combination,emotionally and economically acceptable pregnancy rates wereachieved: 24% per aspiration, 43% per embryo transfer, and 25%per couple. One twin pregnancy resulting in the birth of twohealthy female infants and one ongoing twin pregnancy were achievedwith epididymal spermatozoa; four pregnancies (one twin, twosingletons, one abortion) were achieved with donor spermatozoa.