Tests de Selection des Spermatozoïdes avant Assistance Médicale à la Procréation

There are many procedures to select spermatozoa but those procedures are often specific of each andrology centre. This review that is not an exhaustive review of the numerous methods published is rather devoided to focus on the main techniques, the sperm washing, the filtration methods («SpermPrep»), the swim-up, and the discontinuous density gradients with colloïdal solutions («Percoll», PureSperm” and «Isolate») including iodinated organic molecule as ‘OptiPrep». The first part presents a synthesis of literature in order to bring out standardized procedures, and a summary of the advantages and disadvantages of each step of these procedures. Some specific anomalies of sperm need particular treatments. There is a consensus on the validated techniques to prepare semen samples in the presence of antisperm antibodies, leukocytes, bacterial contaminants, and also semen samples exhibiting hyperviscosity or in case of retrograde ejaculation. Cryopreserved sperm preparation is also described with references to experimental results in freezing-thawing human spermatozoa. The second part proposes guidelines for the therapeutic choice of the appropriate assisted reproductive technologies (i.e.: intra uterine artificial insemination, in vitro fertilization or intracytoplasmic sperm injection) according to semen parameters, and to the outcome of sperm selection and survival.     

Empreinte parentale et Aide Médicale à la Procréation (AMP)

Medical intervention in procreation is not recent, as the first artificial insemination (AI) was performed more than two centuries ago. However, the interference in the reproductive process with Al is limited. The first major change concerned the possibility of fertilizing oocytesin vitro (IVF) and culture of preimplantation embryos before their transfer to the uterus. In the early nineties, it was shown that direct injection of a single spermatozoon, even abnormal or immature, into an oocyte could result in a viable embryo and child. These techniques expanded very rapidly and 45,000 IVFs, with ICSI in 50% of cases, were performed in France in 2001 (FIVNAT). Although a high incidence of major defects has not been reported, the health status of children born by these techniques is a growing concern. Congenital malformations [Hansenet al., 2002], chromosomal abnormalities [Van Steirteghemet al., 2002], neurological disorders [Stromberget al., 2002] and low birth weight [Schieveet al., 2002] have been observed and discussed, but none of them seems to be statistically much more frequent after assisted reproductive technology (ART). It is important to determine the mechanism of these defects in order to prevent them. These risks may be related to the parents’ health status and to their infertility, but they could also be linked to the techniques used for procreation. Recently, several human and animal studies have suggested an increased risk of imprinting disorders in ART offspring [Debaunet al., 2003; Gicquelet al., 2003; Maheret al., 2003; Hallidayet al., 2004]. Several elements can be considered to be responsible for these defects and each step of reproductive technology could be concerned and must be studied. Priority should be given to confirm the incidence of rare genomic imprinting diseases, such as Beckwith Wiedemann Syndrome and Angelman Syndrome after ART. Should systematic analysis of the methylation status of several imprinted genes therefore be performed to evaluate the respective influence of the use of immature gametes, ovarian stimulation and embryo culture involved in IVF/ICSI? It would also be important to evaluate other epigenetic modifications to determine the role of epigenetic deregulations that could be related to ART.     

Une forme de tératozoospermie monomorphe: Le syndrome des spermatozoïdes macrocéphales et ses variants

Macronuclear spermatozoa is a rare sperm defect, but always raises problems in the context of assisted reproductive technology (ART). The authors propose a review of the macronuclear spermatozoa syndrome and its phenotypic variants. In practice, ART can be proposed except in the homogeneous form of this sperm defect. Evaluation of chromosomal abnormality rates in the spermatozoa of these patients must be performed, followed by multidisciplinary discussion and clear information of the patients prior to ART.     

Stratégies biologiques à l’heure de l’Assistance Médicale à la Procréation chez le blessé médullaire

Most men with spinal cord injury (SCI) men have fertility problems caused by anejaculation and decreased fertility of the ejaculate. There are two main causes for the impaired reproductive potential in SCI men: ejaculatory dysfunction and poor quality semen. However, current treatment techniques allow a large number of SCI males to achieve ejaculation (rectal electro-stimulation, penile vibrator stimulation). Firstly, masturbation and/or penile vibrator are used at home allowing the couple to perform insemination themselves. The semen of men with spinal cord injuries has commonly been characterised by small volume, abnormal count (low or high), decreased sperm mobility, increased formation of reactive oxygen species, sperm autoimmunity, necrospermia. This impairment is thought to be due to insufficient drainage, genitourinary infections and raised scrotal temperature. Testicular biopsy reveals varying degrees of tubule degeneration and decreased spermatogenetic activity. Semen could be used for various assisted reproductive technologies such as intrauterine insemination,in vitro fertilisation (IVF) and microinsemination (ICSI). The literature reports pregnancy rates by intrauterine insemination of about 15 to 20% per couple. Clinical pregnancy rates after IVF and ICSI techniques are 30% per cycle and these results are comparable to the clinical pregnancy rates when these techniques are performed for female infertility. However, semen must be frozen as soon as possible after the injury and the patient must be informed about the various available assisted reproductive technologies.     

La filiation et l'aide médicale à la procréation

Kinship following artificial insemination by unknown donor defines the child's mother and father as unknown. Although it could have been possible to construct this kinship on the model of adoption, the model adopted was that of blood and organ donation, which completely erases the donor's identity so that the recipient can take complete possession of the donated substance. But is gamete donation the same thing? Gamete donation concerns procreation and the parents' sexuality. Would it be possible to no longer consider the parents' sexuality to be exclusively reproductive sexuality allowing recognition of the progenitors alongside the mother and father?     

Groupe de prise en charge clinico-biologique en Assitance Médicale à la Procréation (AMP) des Patients à Risque Viral

La prise en charge en Assitance Médicale à la Procréation concerne de plus en plus de couples à risque viral. Cette prise en charge est aujourd’hui le seul moyen efficace de réduire une transmission viral mère-enfant ou entre partenaires séro-différents. Elle met en jeu une importante activité pluridisciplinaire où travail en commun et concertation entre les différentes équipes médicales clinico-biologiques sont les deux points clefs.     

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.     

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.     

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.     

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.     

Traçabilité en assistance médicale à la procréation

An ART traceability procedure provides a written record of the controls performed to ensure precise allocation of gametes or embryos during insemination or IVF techniques. This procedure requires analysis of the various steps associated with a risk of error and optimization of safety systems. The author provides a basis for further thought on this subject.     

Estrogènes et spermatozoïdes

Aromatase is the terminal enzyme responsible for estrogen biosynthesis; it is present in the endoplasmic reticulum membrane of steroidogenic cells in vertebrates. This enzyme functions with the ubiquitous reductase as the electron donor. The aromatase gene is unique and its expression is regulated in a tissue and more precisely in a cell-specific fashion via the alternative use of several promoters located in the first exons. This enzymatic complex is generally involved in development, reproduction, sexual differentiation and behaviour, but also in bone and lipid metabolism, brain functions and diseases such as breast and testicular tumors. The aromatase gene expression and its transduction in a fully active protein in testicular somatic cells and germ cells together with the widespread distribution of estrogen receptors (ERα & β) in the testis and the genital tract of the male, are clearly in favor of a physiological role for estrogens in the spermatogenesis processings especialy in sperm maturation. Therefore, we begin to understand the physiopathological roles of the estrogens in males indeed, the aromatase deficiency is associated, with severe bone maturation problems and sterility in man. Conversely, it is also obvious that estrogens in excess are responsible of the impaired spermatogenesis. These female hormones (or the ratio androgens/estrogens) do play a physiological role in the development and maintenance of male gonadal functions and obviously, several steps are concerned especially the sperm production and maturation.     

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.     

Utilisation des spermatozoïdes cryoconservés: fréquence et résultats

Semen cryopreservation has become a major activity of CECOS units. In 2002, 2,323 patients were referred to a CECOS unit for semen cryopreservation prior to radiotherapy or chemotherapy. Cryopreservation of one or several semen specimens was performed for 2,124 patients, which represents a total of more than 60,000 straws per year. The two diseases mainly concerned by cryopreservation prior to treatment with a high risk of sterilization are testicular cancer (about 40% of requests) and lymphomas (about 30% of requests). Specimens are generally stored for several years. The most frequent indications are testicular cancer (40% of the patients) and lymphomas (30% of the patients). Due to the continuing improvement of treatment, the subsequent fertility prognosis of patients has improved over recent years, but often remains difficult or even impossible to predict. An assessment of the effective use of these frozen gametes and the results obtained therefore appeared to be interesting. During 2002, 304 men requested the use of their cryopreserved semen. More than 1,000 straws were thawed and used for insemination (195 insemination cycles, 12 pregnancies obtained) orin vitro fertilization (25 conventional IVF cycles, 8 pregnancies; 257 IVF-ICSI cycles, 57 pregnancies). A retrospective cumulative study conducted in 2001 with the collaboration of 15 of the 23 CECOS units calculated, for each year, among patients in whom cryopreservation could be performed, the percentage of patients who subsequently requested the use of straws between the date of cryopreservation and 2001. This percentage varied between 5% and 10%, depending on the time since freezing. Calculation of the percentage of patients for whom destruction of straws was performed, either at the patient’s request, or because of the patient’s death, was also performed according to the same methodology. The percentage of destruction because of the patient’s death varied between 5% and 9%. The percentage of destruction of straws at the patient’s request was close to or greater than 15%, when the storage time exceeded 3 years. The percentage of patients lost to follow-up remains low in these indications for cryopreservation, ranging between 3% and 6% depending on the year. These data are globally coherent with the data reported in the literature. Although the use of straws is not the most frequent outcome of semen cryopreservation, freezing of gametes must nevertheless always be proposed to patients, as their subsequent fertility often remains difficult to predict. Progress in methods of medically assisted procreation also allow a good chance of pregnancy even when few viable spermatozoa have been preserved.     

La procréation médicalement assistée avec spermatozoïdes immatures: Impératifs cliniques et techniques chirurgicales

The authors report their experience with the use of spermatids in TESE programs where mature spermatozoa could not be isolated from testicular biopsies. The details of the indications for spermatid insemination, the technicity of the procedure and the results are exposed.