[In vitro spermatogenesis new horizon to restore fertility?]. / Spermatogenèse in vitro nouvel horizon pour restaurer la fertilité ?

The survival of the young boy after cancer has considerably progressed in recent years due to the efficiency of chemo/radiotherapy against the tumor cells. However, this treatment causes adverse effects on healthy tissues, including fertility. Freezing testicular tissue before highly gonadotoxic treatment is a prerequisite for preserving fertility in prepubertal boys that do not produce sperm yet. But which strategy proposes to restore fertility from frozen-thawed testicular tissue? One potential solution would be to consider an in vitro maturation of spermatogonial stem cells. In this article we trace the chronological development of in vitro spermatogenesis that resulted in mouse sperm production in vitro and give an overview of new challenges for the future.

The feasibility of fertility preservation in adolescents with Klinefelter syndrome.

STUDY QUESTION: Is fertility preservation feasible after the onset of puberty in adolescents with Klinefelter syndrome (KS)? SUMMARY ANSWER: Fertility preservation counseling should be an integral part of the care of XXY adolescents. Frozen ejaculated or testicular spermatozoa and even frozen immature germ cells can give them the potential to conceive their genetic progeny. However, no biological or clinical parameters were predictive of mature or immature germ cell retrieval. WHAT IS KNOWN ALREADY: KS is the commonest sex chromosome disorder observed in azoospermic infertile males. Testicular sperm extraction success decreases with age and after testosterone therapy. Arguably, spermatozoa should be retrieved from KS males at the onset of puberty and before testosterone therapy to increase the chance of success. STUDY DESIGN, SIZE, DURATION: A retrospective study was performed in eight KS adolescents, aged between 15 and 17 years, who were referred for counseling about their future fertility to the center CECOS (Centre d'Etude et de Conservation des Oeufs et du Sperme humain) at Rouen University Hospital between October 2008 and December 2011. PARTICIPANTS/MATERIALS, SETTING, METHODS: The patients were first seen with their parents and then separately. It was proposed to them that they should provide a semen sample, if this was azoospermic, two other semen samples spaced by 3 months were collected. If azoospermia was confirmed, a bilateral testicular biopsy was proposed for sperm retrieval and testicular tissue preservation. Each adolescent met the psychologist before undergoing testicular biopsy. Paraffin-embedded testicular tissue was evaluated after staining with hematoxylin-eosin and saffron and immunostaining using vimentin, anti-Müllerian hormone, androgen receptor and MAGE-A4 antibodies. Sertoli cell maturity, germ cell identification and lamina propria alteration were assessed on seminiferous tubules. MAIN RESULTS AND THE ROLE OF CHANCE: KS adolescents were not deeply concerned about their future fertility and only became involved in the process of fertility preservation after at least three medical consultations. The parents agreed immediately that fertility preservation should be attempted. Seven non-mosaic XXY adolescents presented with azoospermia and one XXY/XY adolescent had oligozoospermia. Increased plasma levels of FSH and LH as well as bilateral testicular hypotrophy were observed in all patients. The XXY/XY adolescent banked four semen samples before testosterone replacement therapy. Two patients refused testicular biopsy. Five patients accepted a bilateral testicular biopsy. Spermatozoa were retrieved in one patient, elongated spermatids and spermatocytes I in a second patient. LIMITATIONS, REASONS FOR CAUTION: The number of patients enrolled in our study was low because the diagnosis of KS is only rarely made before or at the onset of puberty. Most XXY males are diagnosed in adulthood within the context of male infertility. WIDER IMPLICATIONS OF THE FINDINGS: Spermatozoa can be retrieved in semen sample and in testicular tissue of adolescent Klinefelter patients. Furthermore, the testis may also harbor spermatogonia and incompletely differentiated germ cells. However, the physician should discuss with the patient and his parents over a period of several months before collecting a semen sample and performing bilateral testicular biopsy. Fertility preservation might best be proposed to adolescent Klinefelter patients just after the onset of puberty when it is possible to collect a semen sample and when the patient is able to consider alternative options to achieve fatherhood and also to accept the failure of spermatozoa or immature germ cell retrieval.

Modification of chromosomal architecture in human spermatozoa with large vacuoles.

Human normal spermatozoa present a specific chromatin organization, illustrated particularly by the non-random chromosome positioning. Spermatozoa with large vacuoles, described using motile sperm organelle morphology organization (MSOME), are associated with nuclear alterations, such as abnormal chromatin condensation and aneuploidy. To question a probable association between large nuclear vacuoles and chromatin disorganization, we evaluated chromosomes X, Y and 18 topography in normal spermatozoa (NS) compared with spermatozoa with large vacuoles (SLV). After centrifugation on a gradient density system, 229 NS (spermatozoa presenting a normal nuclear shape and a vacuole area <6.5% of head area) from 10 normal semen samples and 221 SLV (spermatozoa presenting a vacuole area >13% of head area) from 10 semen samples with teratozoospermia were selected using MSOME. A three-colour FISH was carried out using α-satellite centromeric probes for chromosomes X, Y and 18. For each chromosome, longitudinal and spatial positioning of centromeres was analysed. Distribution of each chromosome was non-random in NS and in SLV, whatever the methodology used. Using longitudinal positioning, distribution of chromosome 18 and chromosome Y centromeres did not differ significantly between SLV and NS. On the contrary, chromosome X centromeres were more frequently positioned in the posterior region of sperm nucleus in SLV (p = 0.01). Considering spatial positioning, distributions differed significantly between SN and SLV for chromosome Y (p = 0.02) and chromosome 18 (p < 10(-4) ) and marginally for chromosome X (p = 0.08). Our study concluded to a modification in chromosomes X, Y and 18 centromere topography between NS and SLV, representing a novel and supplementary evidence to argue chromatin disorganization in SLV.

[Male gamete spermatozoon or spermatid?]. / Gamète mâle un spermatozoïde ou une spermatide?

Normal spermatogenesis results from a balance between process of cell proliferation, cell differentiation and apoptosis that concern somatic cells and germ cells. Dysfunction of spermatogenesis may be the result of constitutional or acquired abnormalities of spermatogonia stem cells or somatic cells. To overcome these problems, it seems necessary to implement preventive measures for germ stem cell preservation or substitute measures to replace them, the objective being to replicate in vivo or in vitro the process of spermatozoa production. This article will discuss the different experimental strategies for considering the in vivo or in vitro production of spermatozoa, outside the physiological process.

Relationship between conventional sperm parameters and motile sperm organelle morphology examination (MSOME).

With the motile sperm organelle morphology examination (MSOME), spermatozoa morphology may be assessed directly on motile spermatozoa at high magnification (up to 6600×). This procedure describes more precisely spermatozoa abnormalities, especially head vacuoles. However, no consensus has been established concerning normal or abnormal MSOME criteria. The aim of our study was to define MSOME vacuole criteria assessed objectively with a digital imaging system software to establish a potential relationship between conventional semen parameters. A total of 440 semen samples were obtained from males consulting in Rouen University Hospital Reproductive Biology Laboratory. Conventional semen analysis (volume, sperm concentration, progressive motility, vitality and morphology) and MSOME assessment {sperm head length, width and area as well as vacuole number, vacuole area and relative vacuole area to sperm head [RVA (%) = [vacuole area (µm(2))/head area (µm(2))] × 100)]} were performed for each semen sample. Among our 440 males, 109 presented normal conventional semen parameters and 331 abnormal ones. Sperm head vacuoles were significantly larger in abnormal semen samples (p < 0.0001). RVA was the most discriminative MSOME criterion between normal and abnormal semen samples according to ROC curves analysis, and was negatively correlated with poor sperm morphology (r = -0.53, p < 0.0001). We concluded to (i) the normal occurrence of vacuoles in sperm head whatever the normality or abnormality of semen parameters, (ii) the discriminative function of the RVA to distinguish semen samples with normal and abnormal parameters, and (iii) the strong correlation between high RVA and poor sperm morphology.

Management of male infertility due to congenital bilateral absence of vas deferens should not ignore the diagnosis of cystic fibrosis.

Microsurgical or percutaneous epididymal sperm aspiration and intracytoplasmic sperm injection (ICSI) are proposed to overcome male infertility due to congenital bilateral absence of vas deferens (CBAVD). CBAVD has been associated with mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene and consequently, genetic counselling has to be addressed before beginning ICSI procedure. However, management of male infertility due to CBAVD should not ignore a mild form of cystic fibrosis. We describe the case of cystic fibrosis late diagnosis performed in a 49-year-old infertile men with CBAVD. CFTR molecular testing detected two mutations F508del and A455E corresponding to a cystic fibrosis genotype. Pneumological evaluation revealed a severe obstructive respiratory disease, bronchiectasis and high sweat chloride levels. Symptoms consistent with a cystic fibrosis have to be identified in infertile men with CBAVD before beginning assisted reproductive procedures.

Assessment of freezing procedures for rat immature testicular tissue.

Fertility preservation has been included in the management of childhood cancer treatment. Cryopreservation of immature testicular tissue is the only available solution for pre-pubertal boys. Different freezing protocols have been developed in several species but without a clearly identified procedure. We tried to evaluate several protocols for cryopreservation of rat immature testicular tissue. Twelve different freezing protocols using different (i) cryoprotectant (dimethylsulphoxide [DMSO] or 1,2-propanediol [PROH]), (ii) cryoprotectant concentration (1.5M or 3M), (iii) equilibration time (30 or 60 min), (iv) equilibration temperature (4 °C or room temperature), (v) size of testicular fragment (7.5mg or 15 mg), (vi) package (straws or cryovials), were compared using cord morphological damage evaluation. A testicular tissue piece of 7.5mg cryopreserved in cryovial using 1.5M DMSO, an equilibration time of 30 min at 4 °C showed fewer morphological alterations than the other protocols tested. The selected freezing protocol was able to maintain rat immature testicular tissue architecture, functionality after testicular pieces organotypic culture, and could be proposed in a human application.

Reduced levels of human apoE4 protein in an animal model of cognitive impairment.

The APOE4 allele is the most common genetic determinant for Alzheimer's disease (AD) in the developed world. APOE genotype specific differences in brain apolipoprotein E protein levels have been observed in numerous studies since the discovery of APOE4's link to AD. Since the human apoE4 targeted replacement mice display characteristics of cognitive impairment we sought to determine if reduced levels of apoE might provide one explanation for this impairment. We developed a novel mass spectrometry method to measure apoE protein levels in plasma. Additionally, we developed an ELISA that replicates the mass spectrometry data and enables the rapid quantitation of apoE in plasma, brain and cerebrospinal fluid. We detected a significant decrease in plasma, brain and cerebrospinal fluid apoE levels in the apoE4 mice compared to apoE2 and E3 mice. We also measured a small (∼19%) decrease in brain apoE levels from aged, non-demented APOE4 carriers. Our findings suggest that a fraction of APOE4-linked AD may be due to insufficient levels of functional apoE required to maintain neuronal health.

Assessment of acrosome and nuclear abnormalities in human spermatozoa with large vacuoles.

BACKGROUND: Spermatozoa with large vacuoles (SLV) may have a negative impact on embryo development. The origin of these vacuoles is unknown. We evaluated acrosome and nucleus alterations in isolated SLV, versus unselected spermatozoa. METHODS: We studied 20 patients with teratozoospermia. Spermatozoa from the native semen sample and spermatozoa presenting a vacuole occupying >13.0% total head area, isolated under high magnification (×6600), were assessed. Confocal and transmission electron microscope evaluations were performed on SLV and native sperm, respectively. Acrosome morphology and DNA fragmentation were analysed using proacrosin immunolabelling (monoclonal antibody 4D4) and terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling assay. Chromatin condensation was evaluated with aniline blue staining. Sperm aneuploidy was assessed using fluorescence in situ hybridization. RESULTS: SLV represented 38.0 ± 5.10% of motile spermatozoa obtained after gradient density centrifugation. Vacuoles were mainly in the anterior and median sperm head (45.7 ± 2.90 and 46.1 ± 3.00%, respectively). Abnormal acrosomes were increased in SLV compared with unselected spermatozoa (77.8 ± 2.49 versus 70.6 ± 2.62%; P = 0.014). Microscopic observations showed an exclusively nuclear localization of large vacuoles. Complete DNA fragmentation was higher in native spermatozoa (P < 0.0001) than SLV, while chromatin condensation was altered in SLV (P < 0.0001). Aneuploidy and diploidy rates were increased in SLV (P < 0.0001). CONCLUSIONS: Sperm vacuoles were exclusively nuclear. In our selected teratozoospermic population, aneuploidy and chromatin condensation defects were the main alterations observed in SLV. Based on results from this small sample of spermatozoa, we propose a global impairment of the spermatogenesis process as a common origin of the morphological alterations.

Progressive loss of synaptic integrity in human apolipoprotein E4 targeted replacement mice and attenuation by apolipoprotein E2.

Inheritance of the APOE4 allele is a well established genetic risk factor linked to the development of late onset Alzheimer's disease. As the major lipid transport protein in the central nervous system, apolipoprotein (apo) E plays an important role in the assembly and maintenance of synaptic connections. Our previous work showed that 7 month old human apoE4 targeted replacement (TR) mice displayed significant synaptic deficits in the principal neurons of the lateral amygdala, a region that is critical for memory formation and also one of the primary regions affected in Alzheimer's disease, compared to apoE3 TR mice. In the current study, we determined how age and varying APOE genotype affect synaptic integrity of amygdala neurons by comparing electrophysiological and morphometric properties in C57BL6, apoE knockout, and human apoE3, E4 and E2/4 TR mice at 1 month and 7 months. The apoE4 TR mice exhibited the lowest level of excitatory synaptic activity and dendritic arbor compared to other cohorts at both ages, and became progressively worse by 7 months. In contrast, the apoE3 TR mice exhibited the highest synaptic activity and dendritic arbor of all cohorts at both ages. C57BL6 mice displayed virtually identical synaptic activity to apoE3 TR mice at 1 month; however this activity decreased by 7 months. ApoE knockout mice exhibited a similar synaptic activity profile with apoE4 TR mice at 7 months. Consistent with previous reports that APOE2 confers protection, the apoE4-dependent deficits in excitatory activity were significantly attenuated in apoE2/4 TR mice at both ages. These findings demonstrate that expression of human apoE4 contributes to functional deficits in the amygdala very early in development and may be responsible for altering neuronal circuitry that eventually leads to cognitive and affective disorders later in life.

Rapid screening of cryopreservation protocols for murine prepubertal testicular tissue by histology and PCNA immunostaining.

Numerous parameters have to be tested to identify optimal conditions for prepubertal testicular tissue banking. Our study evaluated 19 different cryopreservation conditions for immature testicular tissue using a rapid screening method. Immature mice testes were cryopreserved using either 1,2-propanediol (PROH) or dimethyl sulfoxide (DMSO) at a concentration of 0.75 or 1.5 M using a controlled slow-cooling rate protocol with (S+) or without seeding (S+). Equilibration was performed either at room temperature or at 4°C for 15 or 30 minutes. Seminiferous cord cryodamage was determined by scoring morphologic alterations. Cell proliferation ability was evaluated using a proliferating cell nuclear antigen (PCNA) antibody. Testes cryopreserved with optimal conditions were grafted into immunodeficient mice. The highest proportions of PCNA-positive nuclei and lowest morphologic alterations were observed with 1.5 M DMSO. Tissues were more altered with 0.75 M DMSO or PROH. Complete germ cell maturation was observed after allografting of testicular pieces previously frozen with 1.5 M DMSO, S+, 30 minutes. The 1.5 M DMSO, S+ or S+ protocol preserved prepubertal mice testicular tissue architecture and germ cell and Sertoli cell proliferation potential. Allografting of thawed testis fragments into immunodeficient mice confirmed that the 1.5 M DMSO, S+, 30 minutes protocol maintained testicular somatic and germ cell functions. Postthaw histologic evaluation and PCNA immunostaining are useful to rapidly test numerous freeze-thaw parameters. They may also be efficient tools to control human prepubertal frozen testis quality, within the context of a clinical application.

[Laser assisted hatching: Rouen University Hospital outcomes]. / Eclosion embryonnaire assistée: évaluation des indications au CHU de Rouen.

OBJECTIVE: Despite technical progress in In Vitro Fertilisation (IVF) procedure, embryo implantation rate remains low. Assisted hatching has been proposed to facilitate natural embryo hatching and implantation. PATIENTS AND METHODS: Our study has evaluated whether laser assisted hatching improves implantation, pregnancy and live birth rates in different cases. We studied retrospectively 143 IVF cycles concerning more than 38 years old women, 166 IVF cycles after two previous implantation failures and 180 frozen-thawed embryo transfers. RESULTS: Population characteristics were comparable in hatched and control groups. Implantation, pregnancy and live birth rates in women more than 38 years old were comparable with or without assisted hatching. Concerning repeated implantation failures, even if implantation, pregnancy and live birth rates were higher in assisted hatching group (FIV or ICSI), the differences were not significant. After frozen-thawed embryo transfers, implantation rate was significantly better with assisted hatching (19.14% vs 8.84% [p=0.02]). DISCUSSION AND CONCLUSION: Assisted hatching improves embryo implantation rate after frozen-thawed embryo transfer.

Fertility preservation in adolescent males: experience over 22 years at Rouen University Hospital.

BACKGROUND: Sperm banking is a suitable procedure to prevent infertility after cancer therapy in male adolescents. We evaluated the feasibility of semen preservation in 156 adolescents aged between 13 and 20 years and then we assessed fertility outcome after treatment. METHODS: Age, urogenital history, indications for cryopreservation, histological diagnosis and semen parameters were recorded. Fertility status after treatment was assessed by a questionnaire addressed to those patients who had utilized sperm storage. Post-treatment semen analysis was performed for 22 patients. RESULTS: Cryopreservation was possible in 88.5% of cases. Azoospermia was detected in 2.6% of the patients at the time of diagnosis. Malignant disease accounted for 84% of our male adolescents. In this type of disease, semen parameters were significantly altered only among patients with metastatic malignant bone tumour. After treatment, nine patients presented azoospermia, five patients achieved pregnancy spontaneously, two achieved it after assisted reproductive technique using fresh ejaculated spermatozoa and one following sperm donation. Three failed with cryopreserved sperm. CONCLUSIONS: Semen cryopreservation is possible for most adolescents and, regardless of disease type, may be a means of preserving fertility prior to gonadotoxic treatment that might impair the spermatogenesis process.

Comparison of conditions for cryopreservation of testicular tissue from immature mice.

BACKGROUND: Cryopreservation of immature testicular tissue could be considered as a major step in fertility preservation for young boys with cancer. In the present study, eight different freezing protocols were evaluated in immature mice testis. METHODS: Testis from six-day-old mice were frozen using either 1,2-propanediol (PROH) or dimethylsulphoxide (DMSO: D) at 1.5 M. Different cooling rate curves were tested: (i) controlled slow protocol with seeding (CS+) or (ii) without seeding (CS-), (iii) controlled rapid protocol and (iv) non-controlled protocol. Cryodamage of seminiferous cords was semi-quantitatively determined, establishing a scoring of alterations. Cell viability and apoptosis induction were assessed on testicular cell suspensions immediately after digestion (D0) and after a 20-h culture period (D1). Cells recovered after digestion of 100 mg tissue and the rate of living and non-apoptotic cells were quantified at D0 and D1. A long-term culture (9 days) of testis pieces was carried out for the protocol offering the best survival. Testosterone production, intratubular cell proliferation and tubule growth were assessed. RESULTS: DMSO produced optimal results in the different cooling rate curves tested when compared with PROH. Optimal results were obtained for the DCS- procedure (P < 0.05). Testosterone production, tubule growth and cell proliferation of post-thaw pieces were similar to fresh samples. CONCLUSIONS: Testis freezing with 1.5 M DMSO in a CS- procedure was found to maintain not only immature testicular tissue architecture, but also viability of testicular cells, endocrine and partial exocrine functions of the testis. Semi-quantitative evaluation of seminiferous cord cryodamage can be effectively used to rapidly screen optimal freezing conditions and as a possible quality control in a human application.

Complex chromosomal rearrangement and intracytoplasmic sperm injection: a case report.

Complex chromosomal rearrangements (CCRs) are rare events in human pathology and are usually considered to induce severe reproductive impairment by disturbing the meiotic process and producing unbalanced gametes responsible for high reproductive risk. One-third of all CCRs are familial and tend to implicate fewer breakpoints and fewer chromosomes than de novo cases. CCRs are rarely transmitted through spermatogenesis and are primarily ascertained by male infertility. We report a familial balanced CCR, with seven breakpoints involving three chromosomes, which was detected prenatally in a female fetus conceived after intracytoplasmic sperm injection (ICSI) in a couple initially thought to be a carrier of a paternal reciprocal translocation involving two chromosomal breakpoints. Fluorescent in-situ hybridization (FISH) was used to elucidate the complexity of this CCR. The karyotype of the female CCR carrier was balanced and determined as 46,XX.ish t(1;4)(q42;q32)(WCP1+, D1Z5+, WCP4+, D1S3738-, D4S2930+; WCP4+, D4Z1+, WCP1+, D4S2930-, D1S3738+), ins(1;11)(q41;q23q24)(WCP1+,WCP11+, D11S2071-, MLL+; WCP11+, D11S2071+, WCP1-, MLL-), ins(4;11)(q23;q14q23)(WCP4+,WCP11+; WCP11+,WCP4-). The same balanced CCR was confirmed in her oligozoospermic father. We report, to our knowledge, the first case of ICSI performed in an infertile male with CCR, resulting in a balanced CCR carrier female with a normal clinical follow-up at 4 years of age. This particular case stresses the point of the relevance and feasibility of ICSI procedure in cases of balanced CCRs.

Chromosome constitution and apoptosis of immature germ cells present in sperm of two 47,XYY infertile males.

BACKGROUND: In order to assess sperm alterations observed in some XYY males, we analysed the chromosome constitution as well as apoptosis expression in germ cells from two oligozoospermic males with high count of immature germ cells in their semen. METHODS: Sex chromosome number and distribution were assessed at pachytene stage by fluorescence in situ hybridization (FISH). Immature germ cells and spermatozoa were examined by FISH and TdT (terminal deoxynucleotidyl transferase)-mediated dUDP nick-end (TUNEL) assay, combined with immunocytochemistry using the proacrosin-specific monoclonal antibody (mAb 4D4). RESULTS: For patients 1 and 2, two Y chromosomes were present in respectively 60.0 and 39.6% of pachytenes. The three sex chromosomes were always in close proximity and partially or totally condensed in a sex body. XYY spermatocytes I escape the pachytene checkpoint and achieve meiosis. Nevertheless, nuclear division and/or cytokinesis were often impaired during meiosis leading to diploid (mainly 47,XYY cells) and tetraploid (94,XXYYYY) meiocytes. The presence of binucleated (23,Y)(24,XY) immature germ cells resulting from cytokinesis failure agree with a preferential segregation of the two Y chromosomes during meiosis I. In addition, 69.6% (patient 1) and 53.12% (patient 2) of post-reductional round germ cells were XY. However, high level of apoptotic round germ cells (94.9% for patient 1 and 93.3% for patient 2) was detected and may explain the moderate increase of hyperhaploid XY spermatozoa. Segregation errors also occurred in the XY cell line responsible for disomic 18 and X, as well as 46,XY diploid spermatozoa. CONCLUSIONS: Our data are in agreement with the persistence of the extra Y chromosome during meiosis in XYY oligozoospermic males responsible for spermatogenesis impairment and a probable elimination via apoptosis of most XYY germ cells not solely during but also after meiosis.

[Cryopreservation of immature testicular tissue]. / Congélation du tissu germinal chez le garçon.

An increased incidence of cancer is observed in the population of adolescents and young adults since thirty years. Major progress in cancer diagnosis and therapy is unfortunately associated to high degree of toxicity on gonad function. Cryopreservation of ovarian tissue is performed in girls and women before cancer treatment with high risk of infertility. Procedures for ejaculated or testicular extracted spermatozoa are well defined. However, for prepubertal boys or after ejaculated sperm collection failure, mature or immature testicular tissue banking should be proposed. Still, an optimal cryopreservation protocol is a prerequisite for clinical application and does not exist for the moment. Furthermore, the future applications of immature testicular tissue banking should be developed, not solely germ cell in vitro maturation but also autologous testicular tissue grafting.

Apolipoprotein E allele-dependent pathogenesis: a model for age-related retinal degeneration.

Age-related macular degeneration (AMD) is a late-onset, multifactorial, neurodegenerative disease of the retina and the leading cause of irreversible vision loss in the elderly in the Western world. We describe here a murine model that combines three known AMD risk factors: advanced age, high fat cholesterol-rich (HF-C) diet, and apolipoprotein E (apoE) genotype. Eyes of aged, targeted replacement mice expressing human apoE2, apoE3, or apoE4 and maintained on a HF-C diet show apoE isoform-dependent pathologies of differential severity. ApoE4 mice are the most severely affected. They develop a constellation of changes that mimic the pathology associated with human AMD. These alterations include diffuse sub-retinal pigment epithelial deposits, drusenoid deposits, thickened Bruch's membrane, and atrophy, hypopigmentation, and hyperpigmentation of the retinal pigment epithelium. In extreme cases, apoE4 mice also develop marked choroidal neovascularization, a hallmark of exudative AMD. Neither age nor HF-C diet alone is sufficient to elicit these changes. We document choroidal neovascularization and other AMD-like ocular pathologies in an animal model that exploits known AMD risk factors. The model is additionally attractive because it is not complicated by invasive experimental intervention. Our findings in this model implicate the human apoE E4 allele as a susceptibility gene for AMD and support the hypothesis that common pathogenic mechanisms may underlie AMD and Alzheimer's disease.

[Chromosome abnormalities of spermatozoa]. / Anomalies chromosomiques du spermatozoïde.

Chromosome meiotic pairing during male meiosis is a major event for chromosome segregation during anaphase I and spermatogenesis normal process. Chromosome non-disjunctions responsible for aneuploidy in male gametes can be observed during the first and the second meiotic divisions. The analysis of sperm nuclei chromosome constitution is a major and indirect tool for assessing male meiotic non-disjunctions and the genesis of chromosomal abnormalities. This evaluation has been performed initially by the human sperm/hamster oocyte fusion assay and more recently by fluorescence in situ hybridisation (FISH). Therefore, male populations with increased risk of aneuploidy for their progeny could be identified before entering an in vitro fertilization procedure, and depending on the potential risk a preimplantation or prenatal genetic diagnosis could be performed. For males with constitutional chromosome abnormalities, a specific genetic counselling could also be proposed.