Perspectives de l’analyse de la mobilité des spermatozoïdes

Several studies have shown a good correlation between sperm motility and fertility though the microscopic evaluation of the percentage of motile sperm is highly subjective by nature. Therefore in the last decade, various objectives methods have been proposed to overcome this problem. Two types of methods were developed: The methods based on the analysis of images obtained by microphotography, microcinematography and microvideography and the global, undirect methods based on physical principles. Several systems based on video and image analysis (Computer Aided Sperm Analysis, CASA) have been developed and are used in numerous laboratories of reproductive biology. CASA technology offers the possibility to analyse some characteristics of sperm motion which are related to the fertilization potential and to develop new parameters related to some important aspects of sperm behavior such as hyperactivation. However, there is a large amount of interactions between the operator and the CASA machine. CASA instruments are not “ready-to-use” robots: the reliability of CASA depends largely on the expertise and training of the user and the application of standardized procedures and quality control schemes. By contrast, there is only minimal interaction between the operator and the Sperm Quality Anlyser which is a new device measuring and index of sperm motility highly correlated to the concentration of progressively motile sperm. The device uses light passed through a small sample of semen introduced in a capillary tube to detect variations in optical density that result from moving particles. The reproducibility of the measurements is excellent, the device is easy to use and this is a potentially useful tool for field-work studies. Further investigations of this device in the managment of male infertility is warranted. Finally, both types of objectives approaches are complementary to the conventional analysis of sperm motility and they will not replace it. Standardized procedures have been proposed by the World Health Organization for the subjective evaluation of sperm motility. Such procedures are very useful to reduce significantly the intra- and interlaboratory variations but internal and external quality controls schemes indicate that they are not sufficient to achieve acceptable levels of variation and regular quality controls followed by the definition and the application of corrective procedures are required.     

La vitalité des spermatozoïdes

Most of the numerous techniques used to assess sperm viability only have research applications, while only two classical tests, i.e. eosin-Y and hypo-osmotic swelling test (HOST), are currently used in routine sperm analysis to determine the percentage of viable sperm. A viability rate below 50% of living sperm defines necrozoospermia, a condition whose clinical significance is fairly difficult to assess as the mechanisms of sperm cell death are still poorly understood. However, even when a precise cause for necrozoospermia cannot be identified, abnormal viability requires further andrological investigations with particular emphasis on clinical and laboratory signs of chronic infection of the male reproductive tract. Intracytoplasmic sperm injection (ICSI) can yield very good pregnancy rates, even in couples with the most severe forms of male infertility. However, when no motile sperm are available after sperm preparation, the outcome of ICSI is seriously impaired, probably because of a high risk of injecting dead sperm. In these patients, sperm viability could therefore be assessed by the hypo-osmotic swelling test in order to select only viable sperm for ICSI. However, the long incubation time of sperm in the hypo-osmotic solution, as recommended in the classical HOST procedure, has been shown to be detrimental to the spermatozoa. A single sperm test able to assess the viability of each individual spermatozoon within microdroplets covered by mineral oil therefore seems to be preferable. This selection procedure is less suitable in the case of immotile frozen-thawed sperm, as viability does not appear to be reliably predicted by HOST in cryopreserved sperm. Examination of sperm viability now also evaluates programmed cell death or apoptosis, as apoptotic alterations can be detected in spermatozoa by several techniques. The percentage of apoptotic sperm is correlated with deficient sperm parameters and poor outcome of assisted reproductive techniques. More effective selection procedures are therefore needed in order to identify spermatozoa not only with intact membranes but also with an intact genome to be used for ICSI.     

La majorité des couples procréant par don de sperme envisagent d’informer l’enfant de son mode de conception, mais la plupart souhaitent le maintien de l’anonymat du donneur

Semen donation is anonymous by law since 1994 in France but has been abolished in various countries. We present the results of a study that has been conducted in 14 Cecos in 2006, including 534 couples who were waiting for the assisted procreation, were under treatment, or had already at least one child with donor semen. The results were very similar between men and women and in the various groups. Over 90% of the men and the women are in agreement with donors’ anonymity and less than 10% would like the law to be changed on this point. Approximately 25% of them would give up their parental project if the law was going to change. Almost one-third would like information on the semen donor, mainly on his health, to be transmitted to themselves and to the children. The couples who plan to become parents through semen donation make a clear distinction between donor anonymity and child disclosure on its conception circumstances.     

Capacité des spermatozoïdes à se fixer sur la zone pellucide

After liberation from the seminiferous epithelium, the spermatozoa (SPZ), undergo in the epididymis a serie of functional and metabolic modifications resulting the capacity to ensure fertilization. Fertilization is the fundamental process in sexual reproduction as it permits the initiation and the formation of a new being by the fusion of two germinal cells: the male gamete (spermatozoa) and the female gamete (oocyte). For fertilization to occur the SPZ must recognize the zona pellucida (ZP), bind to it, penetrate it and fuse with the oocyte plasma membrane. Sperm binding to the ZP is an early, crucial event leading to fertilization and pre-embryo development. In mammals, sperm-ZP binding follows a serie of steps that occur in a well-defined chronological order: a) A loose association between SPZ and ZP referred to as «attachment». This shortlived interaction is heterospecific. b) Attachment is followed by a more distinct and persistent association of SPZ with ZP, thus called «binding». This sperm-zona interaction is species-specific, irreversible and mediated by complementary receptors present on the SPZ head and the ZP. c) The bound SPZ then undergoes the acrosome reaction (AR). Which involves fusion and vesiculation of the SPZ outer acrosomal membrane and plasma membrane leading to the release of acrosomal contents and the exposure of the inner acrosomal membrane. This AR is essential for SPZ passage through the ZP and to access to the oocyte plasma membrane where gamete fusion occurs.     

Comment identifier le spermatozoïde vivant?

Testicular sperm extraction (TESE) has been used to retrieve spermatozoa in patients with secretory azoospermia for intracytoplasmic sperm injection (ICSI). However, testicular spermatozoa have poor motility that significantly decreases after cryopreservation and thawing. The major difficulty with testicular spermatozoa is to distinguish between living and dead spermatozoa, as most spermatozoa are immotile. The aim of this study was firstly to report the various methods used to explore spermatozoa vitality. Most tests assess the functional and structural integrity of the sperm membrane, such as staining methods and hypo-osmotic swelling test (HOS-test). We then evaluates the potential of pentoxifylline (PTX), a phosphodiesterase inhibitor of the methylxanthine group, to improve the distinction between living and dead immotile testicular spermatozoa by increasing the number of post-thawed motile spermatozoa. We also analysed the results of 100 ICSI cycles performed with frozen-thawed testicular (n=72) and epididymal (n=28) spermatozoa treated with 3.5 mM PTX. To test the effect of PTX on motility, 14 samples of frozen-thawed testicular spermatozoa from eight patients with secretory azoospermia and six patients with excretory azoospermia were divided into three equal samples: one sample treated with 3.5 mM PTX, one sample initially migrated on two-layer Percoll gradient and then divided into two aliquots (one treated with 3.5 mM PTX, one without treatment), and the last sample without migration and without PTX treatment. The number of motile spermatozoa was evaluated in 10 μL of each sample with an inverted microscope at 15, 30, 60, 120 minutes and 24 hours. We also compared the outcome of ICSI in 100 cycles using frozen-thawed epididymal or testicular spermatozoa between secretory and excretory patients. PTX significantly increased the number of motile frozen-thawed testicular spermatozoa in secretory and excretory azoospermia. In excretory azoospermia, the number of motile spermatozoa was further increased when PTX was associated with migration on Percoll gradient, while PTX alone gave the best results in secretory azoospermia. Fertilization and pregnancy rates as well as embryo quality and division stages were comparable in the two groups. By increasing the number of motile frozen-thawed testicular spermatozoa, PTX improves the selection of living spermatozoa.     

Évolution de la composante lipidique de la membrane plasmique des spermatozoïdes durant la maturation épididymaire

One aspect of mammalian post-testicular sperm maturation is the progressive change in their plasma membrane lipid composition. These modifications in lipids allow sperm cells to fuse with oocytes during fertilization. A significant share of these sperm lipid changes occurs during their descent through the epididymal tubule. It then continues within the female genital tract during the capacitation process, an essential prerequisite for acrosomic reaction and hence fertilization. This review presents what is known concerning the sperm plasma membrane lipid changes during epididymal maturation in various mammalian models. In the first section, after a brief presentation of the classic eukaryotic cell plasma membrane lipid organization, the emphasis is on the particularities of sperm plasma membrane lipids. The second section presents the different changes occurring in the three major classes of lipids (i.e. phospholipids, sterols and fatty acids) during the sperm’s epididymal descent. The final section briefly describes the mechanisms by which these lipid changes might happen in the epididymal lumen environment. The role played by lipid-rich vesicles secreted by the epididymal epithelium via apocrine secretory processes is highlighted.     

Intérêt de l’analyse de la morphologie fine et de la qualité nucléaire des spermatozoïdes dans le cadre des techniques d’AMP

Routine semen examination does not identify minor malformations of the sperm nucleus and chromatin architectural defects, which may be associated with ART outcome and cannot be detected by the embryologist even at 1000x magnification. Recent publications have demonstrated the advantages, compared to routine analysis, of a new method of real-time detailed morphological evaluation of motile spermatozoa: motile sperm organellar morphology examination (MSOME). MSOME is performed with an inverted light microscope equipped with high-power differential interference contrast optics enhanced by digital imaging to achieve a magnification of 10000x. To be considered morphologically normal, a sperm nucleus must have both a normal shape and a normal chromatin content. The aim of the present study was to combine MSOME and sperm DNA fragmentation characteristics to assess reproductive outcome. The study population consisted of the male partners of 52 couples referred for conventional IVF or split cycles (half IVF-half ICSI cycles) and exhibiting normal routine sperm parameters. Spermatozoa were analysed by examining the fine nuclear morphology and DNA integrity using the sperm chromatin dispersion test (SCD test), based on the principle that the deproteinized nuclei of spermatozoa with nonfragmented DNA show extended halos of DNA dispersion that are either absent or only minimally present in sperm nuclei with fragmented DNA. Fertilization rates were significantly lower in the group showing less than 8% of normal spermatozoa according to MSOME criteria, but early embryo development was not affected. Fine sperm morphology correlated with DNA fragmentation rate. These results demonstrate that the assessment of sperm nuclear normality by MSOME analysis and SCD test improves characterization of the semen sample and should be evaluated as a tool for allocating patients to specific assisted reproduction treatments.     

Le chimiotactisme des spermatozoïdes a-t-il un rôle dans la fécondation?

Sperm chemotaxis to follicular fluid has been established by a variety of means in human and mouse spermatozoa. It was found that only a small fraction of a given sperm population (averaging around 10%) is chemotactically responsive and that this fraction constitutes capacitated (ripe) spermatozoa. Both the chemotactic responsiveness and the capacitated state are transient (with a lifetime between 50 min and 4 h) and they occur only once in the sperm’s lifetime. It has been proposed that the role of sperm chemotaxis in mammals (at least in man) is selective recruitment of capacitated spermatozoa for fertilizing the egg, and that the role of the continuous replacement of chemotactic/capacitated spermatozoa is to prolong the duration of time over which capacitated spermatozoa would be available in the female reproductive tract. The sperm chemoattractants have not been identified but they appear to be heat-stable peptides. Thein vivo location of sperm chemotaxis is not known; a number of possible locations are discussed.     

L'excursion dans le Nord et l'Ouest de la France de la société internationale de phytosociologie

Sans résumé     

Syndrome de Klinefelter et microinjections intraovocytaires de spermatozoïdes (revue de la littérature)

Historically, Klinefelter’s is the typical clinical form of secretory azoospermia with no hope of achieving biological paternity. However, ICSI, either from ejaculated sperm or following testicular sperm extraction, have been recently applied to patients with Klinefelter’s syndrome. Papers published until June 2002 have reported microinjections with ejaculated sperm in 9 cases of Klinefelter’s syndrome with extreme oligospermia with the following overall results: 79 mature oocytes, 52 fertilized oocytes, 31 embryos, 18 transferred embryos, 6 pregnancies, 5 births, or from testicular spermatozoa, in 93 cases, with the following results: 347 oocytes, 193 fertilized oocytes, 149 embryos, 78 embryos transferred out of 37 cycles, 24 clinical pregnancies and 2 positive pregnancy tests, and 32 births. Although a publication bias was very likely (successful attempts were published, failed attempts were probably not published), these results were unexpected based on the traditional view on Klinefelter’s syndrome. The rate of aneuploid spermatozoa also appeared to be lower than expected. The real proportion of Klinefelter patients in whom spermatozoa with a good potential for embryonic development can be retrieved and the means of identifying these patients remain to be established.     

Composition lipidique membranaire durant la préparation de spermatozoïdes à la fécondation

The final modifications that the spermatozoa undergo correspond with the destabilization of their plasma membrane. This indispensable step facilitates the fusion of membranes and primes the signal transduction during fertilization. This destabilization is composed of a series of changes and modulation of the lipids in membranes such as cholestérol, phospholipids and glycolipids. Several differences exist in the lipid composition of the plasma, acrosome, nuclear and mitochondrial membranes of spermatozoa. The principal membrane phospholipids are phosphatidyl choline, phosphatidyl ethanolamine and sphingomyelin. Plasma membrane of sperm is also rich in polyunsaturated fatty acids (PUFA) linked to phospholipids. Such as C₁₈∶2n?6, C₂₀∶4n?6 and large amounts of docosahexaenoic acid (C₂₂∶6n?6). The amount of membrane lipids in human sperm varies considerably between patients. This variation, could influence certain functional properties of the sperm cells such as their ability to undergo capacitation, the acrosome reaction and the fusion between sperm and oocyte membranes. The lipid composition of the human sperm cell can be altered during the process of freezing-thawing. A significant decrease in phospholipids (phosphatidyl choline, phosphatidyl ethanolamine), and PUFA in particular docosahexaenoic acid and arachidonic acid was observed. Human spermatozoa have a molar cholestérol/phopholipid ratio ≤1.0, and reduces during capacitation due to loss of cholestérol. In addition, the decrease in the levels of cholestérol and the methylation of phospholipids is involved in the modification of membrane fluidity and in the maturation of the sperm plasma membrane receptors. Therefore it seems that the methylation is important for the fusion between sperm and oocyte membranes. Intrinsic sperm phospholipase A2 also plays a role in the destabilization of the plasma membrane by producing of lysophospholipid. Therefore this enzyme and free fatty acids are believed to play a role in the acrosome reaction, an indispensable event facilitating the fusion between sperm and oocyte membranes.     

L’analyse automatisée de la morphologie du spermatozoïde

Examination of sperm morphology is one factor of evaluation of sperm function, but it can also be considered as a biomarker of testicular function. All publications showed a high variability in observed results, in relation with different methods of staining slides and classifying sperm morphology, and a large subjectivity in the visual assessment. Automated sperm morphology analysis (ASMA) have the potential to provide more objective, accurate, and precise morphometric measurements of spermatozoa. Standardisation of the methods of slides preparation is first essential. Analysis of the sperm head morphometry appears the more accessible for the ASMA and could give selective parameters in the evaluation of fertility, in complement with motion sperm analysis. In the other hand automated analysis of all sperm abnormalities appears illusory with actual instruments, because the midpiece or the flagellum is a little structure weakly stained, and thus difficult to be identified by the computer. Until more rigorous and consistent definitions of sperm features can be developped, in relation with testicular function, the pronostic value of existing sperm abnormalities classifications is limited.     

Données descriptives de l’activité d’Assistance médicale à la procréation avec don de spermatozoïdes au sein des CECOS de 1973 à 2006 en France

Introduction

The first CECOS (Centre for study and conservation of human eggs and sperm) was created in 1973 by Georges David and till today, most of the activity of sperm donation in France is managed by the CECOS. This work presents a detailed report of the activity of sperm donation between 1973 and 2006 in this French CECOS network.

Material and methods

Annual activity reports have been compiled by the French CECOS network since 1973. We have collected and analysed these annual reports in order to establish a general estimation of the activity of sperm donation in France during the period of more than 30 years.

Results

Sixty-nine thousand nine hundred forty-five couples asked for assisted reproductive techniques with sperm donation (mainly artificial insemination) to conceive their first child. About 20% of these couples tried to conceive a second or third child. A total of 44,045 children were thus conceived with the effective contribution of 10,347 donors of spermatozoa (out of 16,971 donors who came in the centres for a donation). This report of activity is the largest ever published.     

Viols, sperme, spermatozoïdes, identification et paternité médicolégale

The 3 main objectives of DNA analysis in forensic cases are: first, to establish the genetic profile of an evidence sample (the present study deals with semen stains); second, to identify suspects by comparison with the evidence sample genotype; and third, to identify the biological father of a foetus or child by paternity testing. These tests are very strictly controlled in France. Clinicians must be aware of the technical specificities and requirements to avoid interfering with subsequent analysis and/or use of the data in court.     

Indications de la recherche des anticorps anti-spermatozoïdes

The clinical significance of antisperm antibodies (ASA) is highly controversial. A significant percentage of infertile men and women present immunity to spermatozoa, suggesting that ASA may interfere with the fertilizing capacity. ASA can act negatively on sperm parameters, sperm-cervical mucus interaction, gamete fusion and possibly also on the first step of embryonic development. ASA are present in approximately 2.8% to 26% of the male population and 0.2% to 1.6% of women. The pathogenesis of immunity to spermatozoa had not been fully elucidated: breakdown of normal protective mechanisms, i.e. blood-testis barrier, or epithelial barrier in women, and other mechanisms of immunological sperm tolerance, such as regulation of suppressor T lymphocytes. The indication for antisperm antibody testing is based on clinical and laboratory findings of infertile patients. In men, indictions for ASA testing include a history of genital disease, surgery for genital abnormalities, vasectomy, obstruction or injuries of the male genital tract, infection of accessory glands, long-standing infertility, alteration of semen parameters (agglutination, motility), mucus penetration, and reduced fertilizing capacity in IVF. In many cases, no etiological cause of autoimmunity is found and a genetic predisposition has been suggested. A majority of women do not develop antisperm antibodies, despite repeated contact with spermatozoa during their sexual life. Upper genital tract infection is the main cause of isoimmunization in females, although sexual practices, endometriosis, surgery for cervical neoplasia, recurrent spontaneous abortion and long-term infertility may also be involved. Sperm-cervical mucus impairment is the most obvious effect of immunization in women associated with IVF failure. Autoantibodies are frequently associated with antisperm antibodies. One of the consequences of the success of ICSI has been a decreased research effort to further the understanding of the origin and relevance of antisperm antibodies and specific antibody-antigen interactions. A better understanding of the natural history of immunological infertility would be useful for patient conseiling and to develop the most effective, efficient and safest management strategies. Such data could also be useful for the development of new tests and immunological methods of male contraception.