Danielle Feneux

Measurement of telomere length on tissue sections using quantitative fluorescence in situ hybridization (Q-FISH)

Loss of telomere repeat sequences occurs after each cell division and telomere shortening has been implicated in cellular senescence. The measurement of telomere length might therefore assess the lifespan of a cell. The aim of this study was to set up and validate a technique enabling the assessment of telomere length on tissue sections. Quantitative fluorescence in situ hybridization (Q‐FISH) with telomeric probes was performed on smears and sections from cell preparations or human tissues. The mean fluorescence intensity of telomere spots (FI/spot) was automatically quantified by image analysis. Telomeric restriction fragment (TRF) length was assessed by Southern blotting. There was a positive significant correlation between telomere length, as assessed by Q‐FISH, and TRF length determined by Southern blotting in corresponding samples (p < 0.01, r = 0.6 for tissue and p < 0.01, r = 0.79 for cells). FI/spot was higher on smears than on sections, but pairwise comparison showed a significant correlation both for cells and for tissues (r = 0.77, p < 0.001 for cells and p ≤ 0.01, r = 0.64 for tissue). Finally, since telomere length is expected to shorten with age, FI/spot was assessed in liver samples according to the age of patients: a negative correlation was demonstrated (r = 0.76, p < 0.01). Inter‐assay variation was 7% for Q‐FISH performed on tissue sections and 12% on touch preparations. This study shows that Q‐FISH can be performed with confidence on fixed frozen tissue sections in order to assess telomere length. It is an easy, accurate, and reproducible in situ method for assessing telomeres in the context of cell type and tissue architecture. Copyright

Effect of sperm-associated antibodies on the dynamics of sperm movement and on the acrosome reaction of human spermatozoa

Anti-sperm antibodies were eluted from the sperm cell fraction of autoimmune human ejaculates and transferred onto normal motile spermatozoa. The movement and the acrosomal status of these antibody-coated spermatozoa were evaluated after incubation in a capacitating medium. The amplitude of lateral head displacement (ALH) and the straight line velocity (VSL) were analyzed using an HTM automated motility analyser. Acrosomal loss was monitored by an FITC-conjugated lectin binding technique. During the 6-h incubation in BWW-BSA medium, antibody-free and antibody-coated spermatozoa exhibited significant changes of ALH and VSL distribution that evolved differently in the two populations. The dynamics of sperm movement in control spermatozoa were apparently modified by the presence of antibodies on the sperm membrane. The low percentage of spontaneous acrosomal loss obtained in control populations, even after 20 h of incubation, was not modified by the presence of antibodies on spermatozoa. However, the same antibodies decreased the acrosomal loss induced by a calcium ionophore after 3 h of incubation in capacitating conditions. These results suggest that sperm capacitation and acrosome reaction, considered as essential for successful fertilization, can be altered by antisperm antibodies present on human ejaculated spermatozoa.

Congenital anerythremic erythroleukemia presenting as hepatic failure

We report an atypical case of congenital erythroleukemia in a child born with hepatosplenomegaly and abnormal liver tests. The initial peripheral blood cell count showed anemia and hyperleukocytosis with erythroblastosis that disappeared 1 week later. During the next 5 weeks, no blasts were found in the blood, and less than 5% were found on 2 successive bone marrow aspirates. The infant died of hepatic failure. The suspected diagnosis on a premortem liver biopsy was confirmed by an autopsy that showed a blastic infiltration in many organs. These cells expressed only erythroid markers glycophorin A and C. Rearrangement of the myeloid lymphoid leukemia gene was not found by fluorescence in situ hybridization. The main differential diagnoses include metabolic diseases, Langerhans histiocytosis, Pepper syndrome, transient myeloproliferative disorder, and leukemoid reactions. Although some of these can be excluded by the pathologist, others require a multidisciplinary confrontation: clinical, biologic, genetic, and pathologic examinations.

Fécondance in vivo et in vitro de spermatozoïdes épididymaires humains immatures

In cases of congenital absence of vas deferens (9 patients) or after failure of previous epididymovasostomy (2 patients), in vitro fertilization (IVF) was attempted with spermatozoa surgically obtained at the epididymal caput level. These sperm populations showed little progressive motility (5.9 +/- 6.5%) and an marked necrozoospermia (19.3 +/- 17.4%). Stimulation by caffeine (4.5 mM) alone or associated with heterologue normal seminal fluid resulted in most of the cases in an initiation of motility with an improvement of the progressive velocity. In 9 IVF attempts, 31 mature oocytes were inseminated with 5.10(3) to 1.5.10(6) motile spermatozoa. The dynamic characteristics in 3 inseminated sperm populations were Vp (24.2 +/- 8.3 microns/s), Ah (8.6 +/- 2.0 microns) at room temperature. Sperm binding to zona pellucida was decreased (0 to about 20 spermatozoa per oocyte) and there was no fertilization. In the same period, 21 attempts of intra uterine insemination and 14 attempts of intracervical inseminations were made in 5 couples who remained infertile after patent high epididymovasostomy (4) or vasovasostomy (1) and having immature spermatozoa stimulated as previously described. Antisperm antibodies were detected on the ejaculated spermatozoa in four men. No pregnancy was obtained with these immature stimulated spermatozoa. The fertility of the female partners was confirmed in 3 women after insemination with donor sperm.

Pregnancy after subzonal insemination with spermatozoa lacking outer dynein arms

The absence of outer dynein arms in the sperm flagellum induces an abnormal movement pattern associated with male infertility. These spermatozoa can decondense in zona-free hamster oocytes but result in a very low fertilization rate in in vitro fertilization. We hypothesized that subzonal insemination could help achieve fertilization and pregnancy. A randomized prospective trial (five couples, five cycles) comparing subzonal insemination (n = 31 oocytes) and routine IVF (n = 23 oocytes) was carried out. Oocytes were microinjected with 8.5 +/- 3.6 spermatozoa. In a second series (nine cycles), all the oocytes were microinjected with 10.5 +/- 4.3 spermatozoa. In the randomized series, the fertilization rate was 16.1% without polyploidy, whereas no fertilization was obtained after control IVF insemination. In the second series involving nine couples, six of whom were included in the first series, the fertilization rate increased to 57.8% with a 27.8% polyspermic rate. Eighty-eight per cent of the zygotes cleaved normally (29 out of 33). A total of 11 embryo transfers resulted in three pregnancies, one of which terminated one month later, a second being ongoing and the third delivering a healthy girl. A 21.4% pregnancy rate per cycle, with a 37.5% pregnancy rate per couple, justifies the use of subzonal insemination to treat this particular flagellar dyskinesia.

Interet de la microinjection de spermatozoides sous la zone pellucide pour le traitement de certaines sterilites masculines

Sub-zonal insemination (SUZI) was evaluated, in a randomized prospective study, as a treatment for certain specific types of male factor infertility. Patients were classified according to the results of an extensive semen analysis as being either IVF failure with either normal or subnormal semen quality, specific (e.g. absent outer dynein arms) or non-specific flagellar dyskinesia, or oligoasthenoteratozoospermia. SUZI was found to be an efficient technique for achieving fertilization and pregnancy for all these indications, while there was a total failure of fertilization in the control, in-vitro inseminated oocytes. A total of 395 oocytes were microinjected and 133 fertilized, among which 75 (19% overall) were diploid. More than 81% of these zygotes cleaved normally, and 31 embryo transfers resulted in seven pregnancies. However, results varied widely between the various indications, suggesting that the underlying pathology responsible for the sterility may influence gamete interaction and early embryonic development independantly of the SUZI technique.ResumeParmi les méthodes de microfécondation, la microinjection de spermatozoïdes sous la zone pellucide (SUZI) a été testée au cours d’une étude prospective et randomisée pour différentes pathologies responsables de stérilités d’origine masculine. Les résultats de ces études ainsi que des premières séries de patients inclus dans ce programme sont présentés. Nous distinguons, les échecs de FIV à sperme normal ou subnormal, les dyskinésies flagellaires d’origine structurale comme les absence de bras externes de dynéine ou non spécifiques. Enfin une petite série concerne les oligoasthénotératospermies. Dix neuf pour cent des ovocytes ont été fécondés normalement, et sept grossesses ont été obtenues à partir de 31 transferts d’embryons. Les résultats sont cependant très variables d’une indication à l’autre.

Sliding spermatozoa: a dyskinesia responsible for human infertility?**Supported in part by grant 83C 0668 from Ministere de la Recherche et l’Industrie.

Microcinematographic analysis at 50 frames/second of motile spermatozoa from four sterile men, which were permanently unable to penetrate the cervical mucus or migrate through it was performed at ambient temperature. In all cases, we found the same abnormal pattern of movement, characterized by a very small amplitude of the periodic lateral displacement of the head and abnormal displacement of the wave along the flagellum. The results assessed the importance of normal sperm flagellar dynamics for fertility.