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.     

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.     

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.     

Morphologie des spermatozoïdes

Spermatozoa morphology is one of the qualitative characteristics of spermatogenesis. However, because of both the variations in the definition of normal morphology and the existence of different kinds of sperm abnormalities as well as the use of various techniques of morphology assessment, such a parameter is poorly used in usual laboratory work. Morphological sperm anomalies can be from testicular or post-testicular origines, while the latter is still unproved. The causes of such anomalies are either from genetic origines, but in these cases any spermatozoa demonstrate this anomaly, or due to an endogenous factor with varicocele the most usually quoted but unproved pathology, But exogenous factors, either chemical such as drugs and pesticides or physical such as heat, are also responsible for morphological sperm anomalies. Analysis of sperm morphology is indicative of both the testicular health status (in cases of occupational exposure to chemical or physical toxics) and the fertility potential since morphology is correlated to sperm motility and involved in fertilization through the acrosome reaction.     

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

Sans résumé     

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.     

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.     

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.     

Alcaloïdes de Zanthoxylum decaryi: la decarine,nouvel alcaloide dérivé de la benzophénanthridine

Four alkaloids have been isolated from the stem bark of Zatithoxylum decaryi. Three are known, dictamnine skimmianine, 4-methoxy-1- methyl-2-quinolone. The fourth decarine is new; its structure has been established as 9-methoxy-10-hydroxy-2,3-methylenedioxybenzophenanthridine.¹     

La sélection des spermatozoïdes à fort grossissement permet-elle une diminution de la fréquence des aneuploïdies spermatiques?

Severe male infertility concerns two categories of men. Men with abnormal karyotype, who represent 2 to 14% of infertile men and who can produce sperm cells carrying unbalanced chromosomes related to the patients initial chromosomal reorganization inducing a variable risk of transmission of the abnormality to their conceptus. The second category is men with a normal karyotype but an increased rate of spermatic aneuploidy in a context of severe oligo- and/or asthenozoospermia and men from couples in implantation failure. ICSI is the standard Assisted Medical Reproductive technique for most of these 2 categories despite the obvious increased chromosomal risk. This raises the question of how to morphologically identify sperm cells with abnormal chromosome content during ICSI ? Unfortunately, no relationship has yet been found between sperm morphology in the ICSI sperm fraction (×200) and their chromosome content. Nevertheless, since the end of the 1990s, Bartoov’s team has developed MSOME (Motile Sperm Organelle Morphology Examination) consisting of high-power examination of sperm cells up to × 12,250. This technique was indicated for cases of repeated ICSI failures and appeared to increase pregnancy rates. But was this improvement due to better selection of the chromosomal content of sperm cells to be injected? The present study addressed this question by estimating the value of MSOME in the selection of euploid sperm cells in 2 groups of patients known to have an increased rate of sperm aneuploidy. Group 1 was composed of 2 patients with normal karyotype who presented a macrocephalic sperm syndrome with more than 99% of aneuploid sperm. Group 2 was composed of 11 patients with abnormal karyotype: 6 patients with reciprocal translocation and 5 patients with Robertsonian translocation. The purpose of this study was to compare spermatozoa aneuploidy rates in fresh semen, to those obtained after ICSI selection (×200) and MSOME selection (×6000). Three specific steps of the protocol were (1) all sperm cells selected in MSOME were “top sperm cells“ (2) fixation of selected sperm cell (average loss of 15% during FISH washes) (3) FISH results were validated by two different examiners. FISH analysis of X, Y and 18 chromosomes showed that MSOME eliminates polyploid and diploid sperm cells in patients with macrocephalic sperm syndrome, but the 6 sperm cells selected were all haploid and aneuploid. FISH analysis of X, Y and 18 chromosomes of all other patients did not show any influence of the selection method on the aneuploidy rate. For the 5 subjects with a Robertsonian translocation, the global results of FISH analysis paradoxically showed a significant decrease of the euploidy rate in MSOME selection. The global results of FISH analysis for the 6 patients with mutual reciprocal translocations, showed that the various mutual translocations were not modified between whole sperm and the 2 selection methods. On the other hand, a significant decrease of adjacent 1 and 2 segregation frequency was observed between whole sperm and MSOME selection, associated with a significant increase of 3:1 segregation frequency suggesting that the segregations which modify the structure of chromosomes, for example adjacent 1 and 2 segregations, would induce visible morphological modifications selected by MSOME. We hypothesized that the efficacy of spermatic apoptosis could be modulated by morphology but also by the chromosome contents of the sperm cell. In conclusion, MSOME does not provide any guarantee of the normal chromosome contents of the TOP selected sperm cell. However, these results obtained in a small series of patients suggest that MSOME can eliminate some chromosome abnormalities (adj1 and 2) which would alter sperm nuclear structures.     

Principes de préparation et de congélation des spermatozoïdes testiculaires humains

The advantages and feasibility of human testicular spermatozoa cryoconservation for intracytoplasmic sperm injection (ICSI) have now been clearly demonstrated. However, the freezing protocol is based on empirical knowledge obtained from freezing of ejaculated spermatozoa. Testicular spermatozoa may not be fully mature gametes and may also be retrieved in only limited quantities. Little research has been conducted to determine whether they have the same cryobiological requirements as ejaculated spermatozoa. A better understanding of their cryobiological features and assessment of possible subcellular changes after thawing would help to optimize testicular preparations for cryopreservation (whole biopsies, seminiferous tubules, shredded suspension, single spermatozoa, etc.), freezing-thawing procedure, freezing media, and storage. Finally, there is a growing need for welldefined criteria (nuclear quality, etc.) to evaluate the tolerance of testicular spermatozoa to freezing-thawing procedure for ICSI     

Utilisation des spermatozoïdes testiculaires en ICSI. Intérêt de la culture in vitro. Revue de la littérature

The number of ICSI cycles performed with testicular spermatozoa has increased dramatically over recent years. However, one of the technical limitations of this approach concerns the extremely reduced motility of testicular spermatozoa. However, increased sperm motility was observed after incubating testicular samples for several hours. Therefore, in order to improve ICSI success rates, several authors have tested the effect of previous in vitro culture. We present a review of the literature on this subject. In vitro culture does not appear to be very useful in cases of obstructive azoospermia, as, apart from possible sperm “maturation” during this culture phase, a high proportion of motile spermatozoa is usually already observed prior to in vitro culture. The benefits of in vitro culture appear to be greater in the case of non-obstructive azoospermia, as when spermatozoa are present on the biopsy, they are usually immobile. However, discordant results have been published: after in vitro culture, spermatozoa have been reported to be either motile or mostly dead. Regardless of the type of azoospermia, the best results are obtained after 3–4 days of in vitro culture. Addition of recombinant FSH to the culture medium also appears to be effective. Cryopreservation of testicular biopsies may also be associated with in vitro culture and the in vitro culture/freezing sequence appears to give better results than the freezing/in vitro culture sequence. Very few studies have reported the results of ICSI using frozen in vitro cultured spermatozoa, as most published studies concern fresh spermatozoa, used after 1–2 days of in vitro culture with satisfactory fertilization and pregnancy rates. In vitro culture of testicular spermatozoa may therefore constitute an interesting research approach to improve the results of ICSI when the number of spermatozoa and/or motility are very low. In addition, in vitro culture of testicular spermatozoa appears to be a good tool to study the mechanisms of acquisition of motility, which are still poorly understood.     

L’AQS (Analyseur de la Qualité du Sperme): Evaluation de son intérêt pour l’analyse,la sélection et la congélation des spermatozoïdes

The sperm quality analyzer (SQA) is a device which has been recently proposed to make an objective measurement of human semen quality. It is based on a simple physical method: the measurement of optical density modifications induced by sperm movement. Optical density modification are measured though an electro-optical photoreceptor. Analogical signals produced are transformed in numerical signals and analysed by a microprocessor. The result of the analysis is given in a unique value called «sperm motility index» (SMI). In this study the reproducibility of the results got with the SQA has been measured and the interests of this new diagnostic technique for the andrological laboratory and for human sperm freezing have been evaluated. It can be conclude that SMI measurement is simple, well reproductive and very useful to evaluate the overall quality of a sample. The SMI allows a good prediction of the efficiency of the methods used to select spermatozoa so it can help to choose the best technique, in order to prepare the spermatozoa for a medically assisted procreation. Values of SMI after freezing and thawing of semen samples were also reproducible. They were well correlated to the number of motil sperm per straw as measured by conventional methods, SMI was more objective and accurate. From the value of SMI measured on fresh samples it was possible to predict the freezing tolerance. Therefore measurement of SMI could be very useful to check straws quality and in studies on human semen cryopreservation. Prospective studies are further needed to determine if the SMI could also predict “in vivo” fertility of IVF results and fertilizing ability of frozen thawed sperm.     

Criblage de nouveaux gènes cibles des récepteurs nucléairesdes oxystérols LXRs impliqués dans le maintien de l’épithélium épididymaire et la maturation des spermatozoïdes dans l’épididyme

Liver X receptors (LXRs) are involved in cholesterol homeostasis and lipid metabolism.Ixr knock-out mice for the two isoformsIxra andIxrb exhibit severe disruption of the structure of caput epididymidis segment 1 and 2 epithelium and increased sperm fragility. These defects generate infertility in 10-month-old male mice. The role of LXRs in the epididymis have not yet been investigated. A cell line obtained from mouse caput epididymidis (B2 cells) was used to screen for LXR epididymal target genesin vitro. The presence of one isoform of LXR (LXRα) was detected by immunocytochemistry and the capacity of B2 cells to respond to a synthetic agonist of LXRs (T0901317) was verified. These results validated the use of B2 cells as a model. Bidimensional electrophoresis was performed on B2 cells treated with T0901317. Eight proteins up-regulated by LXRs were isolated. Only one protein has been identified: polyubiquitin, which has already been reported to be involved in cellular cholesterol homeostasis.     

Contribution à la connaissance des bactéries des collections d'eau stagnantes et de leur rôle en hydrobiologie

Ohne ZusammenfassungDirecteur du Centre de Recherches Hydrobiologiques du C.N.R.S.Avec la collaboration technique de Mme L. Goldstein.     

Controle de la multiplication et de la polyploïdie des hepatocytes du rat

Résumé L'injection de substances irritantes de nature chimique variée perturbe les mécanismes qui contrÔlent la multiplication des hépatocytes du rat.Les très jeunes rats réagissent de faÇon intense et reproductible. 15 heures après l'injection il se produit une augmentation brève et intense du nombre des cellules entrant en phase S de sorte que les divisions cellulaires, qui normalement auraient été réparties sur les 30 ou 40 prochaines heures, débutent en l'espace de 2 heures seulement. La croissance hépatique a pris une importante avance qui est suivie par une période de diminution du nombre des mitoses due à une rétroaction inhibitrice.La réaction des rats plus âgés met en évidence un mécanisme inhibiteur permanent qui entraÎne une réponse moins intense et moins régulière.La production d'hépatocytes binucléés et polyploÏdes parait liée à ce mécanisme inhibiteur.La régularité et la sensibilité des réactions des très jeunes rats en fait un matériel de choix pour l'étude des mécanismes régulateurs de la croissance hépatique.

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Control of multiplication and polyploidy of rat hepatocytesA study of the perturbations of physiological regulation induced by injection of irritants

Summary The injection of irritating substances of various chemical nature disturbs the mechanisms regulating the multiplication of rat hepatocytes.The reaction of baby rats is intense and reproducible. It results, 15 hours after the injection, in an increase of the number of cells entering the S phase, so that practically all the divisions which would normally have been scattered over 36 hours are initiated within 2 hours. This burst of synchronized mitosis corresponding to a 36 hours advance in the hepatic growth is followed by a decrease of mitoses due to a feed-back inhibition.The reaction of older rats brings to evidence a permanent inhibitory mechanism that results in a less regular and intensive response.The production of binucleated and polyploÏd hepatocytes seems to be correlated to this inhibitory mechanism.The regularity and sensivity of the baby rat reactions make them a choice material for the study of hepatic growth.