Quality assessment of induced spermatogenesis in hypogonadotrophic hypogonadic men treated with gonadotrophins.

Hypogonadotrophic hypogonadism (HH) is characterized by deficient gonadotrophin secretion, resulting from pituitary or hypothalamic defects. In order to induce spermatogenesis, HH patients are treated with commercially available gonadotrophins. As far as is known, quality and genetic integrity of induced sperm cells have never been investigated, although they represent an important issue, since the ultimate goal of this treatment is to have competent spermatozoa in order to achieve paternity. In order to evaluate the nuclear integrity of induced sperm cells, sperm samples from treated HH patients were compared with sperm samples from normospermic control donors. Sperm cells were analysed by fluorescence in-situ hybridization, using probes specific for chromosomes 13, 21, 18, X and Y, and by TdT (terminal deoxynucleotidyl transferase)-mediated dUDP nick-end labelling assay. Results showed that the rate of aneuploid and diploid sperm cells in patients was not statistically different from controls and that the rate of sperm cells with fragmented DNA was within the normal values. Spermatozoa obtained by gonadotrophin treatment in HH patients are likely to have a balanced chromosomal content and a normal DNA integrity but this conclusion needs to be confirmed by further studies dealing with a greater number of patients.

[Unexpected in vitro fertilization failure in patients with normal sperm: a proteomic analysis]. / Infertilité masculine chez les patients normospermiques : analyse protéomique des spermes normaux non fécondants en fécondation in vitro classique.

OBJECTIVE: Despite normal sperm parameters, 5% of in vitro fertilization (IVF) attempts result in an unpredictable failure of fertilization. In 56% of the cases, there is no obvious oocyte anomaly, but lack of sperm binding to the zona pellucida. This study aims to contribute to clarify the male molecular causes of failures in IVF, which are undetected by classical sperm analysis. PATIENTS AND METHODS: The spermatic proteomic profiles of patients, with a complete failure of fertilization and no spermatozoa bound to the zona pellucida, is compared to controls (patients with normal fertilization and cleavage rates after a classical IVF for tubal indication). All samples are analysed by 2 Dimensional Electrophoresis-Differential In Gel Electrophoresis (2DE-DIGE) after being divided into three fractions according to their isoelectric point (acid, intermediate and basic). RESULTS: Fourteen proteins differentially expressed between all the cases and all the controls were highlighted. Twelve of these proteins were identified by mass spectrometry (six from the acid fraction and six from the basic fraction). Two of these proteins may have an interest in gametic interaction: the laminin receptor LR67 and the L-xylulose reductase. DISCUSSION AND CONCLUSION: More investigation is needed to understand the involvement of the identified proteins in the IVF fertilization failure of the infertile patients in this study.

[Slow freezing and vitrification of human mature and immature oocytes]. / Congélation lente et vitrification des ovocytes humains matures et immatures.

Performance and security questions in human oocyte cryopreservation have been taking researchers for about two decades. Oocytes are usually frozen at metaphase II. Immature oocytes cryopreservation is still a research alternative. Two techniques are currently available for oocyte cryopreservation: slow freezing and vitrification. Experimental data suggest that vitrification has less impact on oocyte physiology than classical slow freezing. After slow freezing of mature oocytes, survival and fertilization rates reach 70 to 80% whereas cleavage rates are around 90%, leading to five implantations and 1.2 births per 100 thawed oocytes. After vitrification of mature oocytes, survival and cleavage rates reach 90% leading to 11 implantations and 1.8 births per 100 thawed oocytes. The obstetrical and neonatal prognosis of these pregnancies is reassuring. No increased risk of congenital anomalies has been observed. However, further evaluation is needed to guarantee the safety of cryopreservation procedures. Immature oocyte cryopreservation is not currently perfected but some indications appear of great interest.

Mutations in the protamine 1 gene associated with male infertility.

In elongating spermatids, human sperm chromatin undergoes a complex compaction in which the transition proteins are extensively replaced by the protamine proteins. Several human studies demonstrate that expression of the protamine proteins is altered in some men with male infertility. For this study, we screened the PRM1 (protamine 1) gene for mutations in a large cohort of 281 men seeking infertility treatment. We identified the c.102G>T transversion that results in an p.Arg34Ser amino acid change in two men. One of these patients presented with oligozoospermia associated with increased sperm DNA fragmentation. The second individual was normospermic but together with his partner sought treatment for idiopathic couple infertility. We also identified a novel missense mutation (c.119G>A, p.Cys40Tyr) in a man with oligoasthenozoospermia. These mutations were not observed in control populations. Interestingly, we also detected variants both 5' and 3' to the PRM1 open-reading frame specifically in infertile individuals. Four individuals with unexplained severe oligozoospermia were heterozygote for a c.-107G>C change that is located at -15 bp from the transcription initiation site of the gene. This mutation may influence PRM1 expression. In addition, a c.*51G>C variant was detected in the 3'UTR of PRM1 specifically in a man with severe oligoasthenozoospermia.

Effects of cryopreservation on the meiotic spindle of human oocytes.

The microtubular meiotic spindle of most mammals, including humans, is very sensitive to cooling [Hum. Reprod. 16 (2001) 2374; Fertil. Steril. 54 (1990) 102; Fertil. Steril. 75 (2001) 769; Zygote 3 (1995) 357] and is rapidly depolymerised even after a slight reduction in temperature to 33 degrees C. Spindle disassembly is dependent on the extent of temperature decrease and its duration. After rewarming, the recovery is far from complete. Cryoprotectants themselves may alter the spindle structure, depending on the duration and temperature of exposure, the duration of recovery at 37 degrees C and the species [Hum. Reprod. Update 2 (1996) 193]. Damage to the meiotic spindle is considered to be the cause of aneuploid embryos, by inducing chromatid non-disjunction and chromosome scattering and by disturbing the sequence of events leading to the completion of meiosis and fertilisation. Nevertheless, a consensus arose from all the studies: appropriate exposure to cryoprotectants and appropriate rates of cooling and thawing allow the cryopreservation of mature oocytes without any significant changes in their second meiotic spindle organisation and without any increase in the rate of aneuploid embryos [Mol. Hum. Reprod. 2 (1996) 445; Hum. Reprod. 8 (1993) 1101; Hum. Reprod. 9 (1994) 684; Microsc. Res. Technol. 27 (1994) 165; Fertil. Steril. 75 (2001) 354]. These fundamental studies in humans, showing good preservation of cell structures after freeze-thaw procedures opened the way to new successful clinical trials with embryos derived from cryopreserved mature oocytes [Fertil. Steril. 68 (1997) 724]. Considering immature oocyte freezing at prophase I (germinal vesicle (GV) stage), a stage which was thought to be less sensitive to cryoinjury, pooled data from the literature showed no advantage in terms of survival rates, fertilisation rates of in vitro matured oocytes and developmental ability of the resulting embryos, especially in unstimulated cycles. Moreover, conflicting results are reported on the effects of freezing on the spindle-chromosome configuration of immature oocytes or in vitro matured oocytes, highlighting the need for large scale studies [Hum. Reprod. 10 (1995) 1816; Hum. Reprod. 13 (Suppl. 3) (1998) 161; Hum. Reprod. 17 (2002) 1885; Microsc. Res. Technol. 27 (1994) 165; Fertil. Steril. 68 (1997) 920]. One child has been born after the use of cryopreserved immature oocytes at GV stage, matured in vitro and fertilised by ICSI [Hum. Reprod. 13 (1998) 3156], demonstrating at least the feasibility of this technique. Improvements are required so as to make mature and immature oocyte cryopreservation an established and safe technique for ART.

Inverse immunostaining pattern for synthesized versus endocytosed alpha-granule proteins in human bone marrow megakaryocytes.

The time of appearance and pattern of expression of several alpha-granule proteins, von Willebrand factor (VWF), fibrinogen and immunoglobulins (Ig) were examined and compared in human bone marrow megakaryocytes (MK) using an immunocytochemical approach. VWF is synthesized by immature MK, whereas it has been shown that fibrinogen is incorporated from the plasma into alpha-granules. The present study was undertaken in order to determine whether there are chronological and morphological differences in the expression of VWF and fibrinogen in vivo in human MK. Seven paraffin-embedded biopsies of normal human bone marrow were labelled with specific antibodies for VWF and for fibrinogen, detected by the alkaline phosphatase anti-alkaline phosphatase (APAAP) method. and analysed by immunomorphometry. We found a clear, statistically significant. difference in the labelling pattern of VWF and fibrinogen. The expression of other endocytosed alpha-granule proteins, immunoglobulins G and A, was therefore studied in bone marrow MK from two patients with multiple myeloma, one with monoclonal IgG and one with monoclonal IgA. The immunostaining pattern was similar to that of fibrinogen and different from VWF, and characteristic of endocytosed alpha-granule proteins. This study demonstrates that: (i) immunohistochemical staining of MK alpha-granules proteins distinguishes the peripheral cockade distribution pattern of endocytosed protein from the perinuclear pattern of endogenously synthesized proteins; (ii) VWF is present in human bone marrow MK when fibrinogen is not yet detectable: (iii) VWF synthesis ceases while fibrinogen is still being incorporated: (iv) immunoglobulins can be detected in MK cytoplasm, with a staining pattern resembling that of fibrinogen.