[Prognostic factors of pregnancy in intra-uterine insemination with sperm of donor: a review of 535 cycles over 7 years]. / Facteurs pronostiques de grossesse en insémination intra-utérine avec sperme de donneur: analyse sur 535 cycles.

AIM: To determine the pronostic factors of pregnancy in intra-uterine insemination using sperm of donor (IIU-D) by analysing female factors, data from the ovarian stimulation and even the characteristics of the donor selected for the recipient couple. MATERIAL AND METHODS: Retrospective study between January 2002 and December 2009. It took place at the University and Hospital of Amiens over 149 couples performing a total of 535 IIU-D cycles. Factors related to the pregnancy were defined thanks to uni and multivariate analysis. RESULTS: Female age was 30.6 ± 4.3 years old (6% of the cycles with women>38). The initiated pregnancy rate per cycle was 27.9%; the birth rate per cycle was 20.4% and the cumulative live birth rate over 63.4% for six cycles. In multivariate analysis prognostic factors of success were: day 3 of estradiol level less than 80 pg/mL (P<0.05), no tube defects (P=0.022), cured and benign womb pathology (P=0.005), a higher number of follicles greater than 16 mm the day of the ovulation triggering (P=0.0018) and a higher number of pregnancies already obtained by the donor in ART in the CECOS (P<0.001). Neither the female age nor the donor age was associated with a probability of success. DISCUSSION AND CONCLUSION: Classical pronostic factors concerning the female profile and the ovarian stimulation quality have been found. But the most interesting and original part is that the most significant factor concerns the donor and his history of pregnancy in Assisted Reproductive Technology (ART) in the center. This may be very useful to guide the choice of the donor for the recipients.

[Pregnancy predictive factors in case of double embryo transfer during "Top Quality" attempts]. / Facteurs prédictifs de grossesses en cas de transfert de deux embryons au cours de tentatives « Top Quality ¼

OBJECTIVES: To identify IVF±ICSI pregnancy predictive factors during "Top Quality" attempts in case of double embryo transfer. PATIENTS AND METHODS: Three years retrospective study (2007, 2008 and 2009) on parameters and results obtained during IVF±ICSI defined as "Top Quality" attempts: first or second attempts on less than 35years old women (age inferior or equal to) with one or two "Top Quality" embryo transfer. RESULTS: In case of double embryo transfer, pregnancy predictive factors are (OR [IC 95%], P): average endometrial thickness on start (4.6 [2.9-5.5], P<0.01), women smoking (4.2 [3.5-4.9], P<0.01), average stimulation duration (3.4 [2.7-3.9], P<0.01), average men age (2.2 [1.7-2.5], P<0.05), gonadotrophins total dose (2.1 [1.1-3.2], P<0.05) and first rank's attempts (1.6 [1.2-2.5], P<0.05). DISCUSSION AND CONCLUSION: Age patient, rank attempts and quality embryo are criteria, which used to guide to a single embryo transfer. Our results incite us to consider other parameters, in particular men age and women smoking status.

[Wrong side iliofascial block in a patient with hip fracture]. / Erreur de côté d'un bloc iliofascial pour une fracture du col fémoral.

We report the case of a patient who received a wrong side iliofascial block immediately before being operated for a femoral neck fracture. This error did not lead to any adverse consequence but this case confirms that wrong side or wrong site error can also occur in anaesthetic practice, especially in emergency procedures, and is not only confined to surgical practice. Anaesthesiologists should be careful when performing unilateral procedures and implement similar strategies than those used by surgeons.

[Professional Practice Evaluation: How to improve quality management in procreation centers?]. / Évaluation des pratiques professionnelles dans un centre d'Assistance médicale à la procréation: comment améliorer la qualité des soins?

OBJECTIVES: The Professional Practice Evaluation (PPE) is at the heart of quality management in procreation centers. Hereby, we report 3 years of EPP in Cytogenetics and Reproduction laboratory in Amiens University Hospital. PATIENTS AND METHODS: This PPE is based upon prospective analysis of in vitro fertilization techniques regarding two major parameters: clinically in improving embryo transfer and biologically by determining fecundation levels. Clinical pregnancies in "Top Quality" trial is chosen as a major indicator of our results. RESULTS: Per transfer, there is an increase of 8% for clinical pregnancies and 31% in "Top quality" trials. DISCUSSION AND CONCLUSION: The improvement in our results allowed us to propose, in favourable conditions, single embryo transfer.

[The bioethics law revision: comparative analysis of contributions from different public and professional offices. Assisted Reproductive Technology, embryo and stem cells research, umbilical cord blood bank]. / La révision de la loi de bioéthique : analyse comparative des contributions de différents organismes publics ou professionnels. Assistance médicale à la procréation, recherche sur l'embryon et les cellules souches, banque de sang du cordon ombilical.

The revision of the bioethics law of 2004 must occur in a five year's time. For this revision, the authorities decided to organize general states of bioethics and requested the production of contributions by the companies, institutions or associations. These texts tackle various subjects, like the Assisted Reproductive Technologies, research on the embryo and the stem cells and the banks of umbilical cord blood. Certain opinions converge, others differ, but all take part in the great debate which will take place at the time of the general conference.

[Premature ovarian failure: which protocols?]. / L'insuffisance ovarienne débutante: quels protocoles?

This review shows the results of the various studies concerning the protocols applied to the women presenting a premature ovarian failure. Will be thus analyzed the natural cycles (or semi-natural), the increase in the dose of gonadotrophins, the clomiphene citrate and the anti-aromatases, the protocols with GnRH agonists long, short, stop or microdoses, the protocols with GnRH antagonists and the adjuvant treatments: aspirin, nitric oxyde, recombinant LH recombining, growth hormone and androgens. The interest of several protocols is to collect a sufficient number of oocytes (and thus of embryos to be transferred), making it possible to obtain reasonable rates of pregnancy. However, it arises that the rates of pregnancy observed among these women depend not only on their ovarian reserve and their age, but are also function of the type of infertility, of the cycle number and the uterus.

[Management of an ovarian stimulation in case of a Kallmann-De Morsier syndrome. The role of LH]. / Prise en charge en induction de l'ovulation d'un cas de syndrome de Kallmann-De Morsier. Réflexions sur le rôle de la LH.

We report a case of ovarian stimulation in a woman with a Kallmann-De Morsier syndrome, which resulted in a triple pregnancy and childbirth by caesarean section at 36 weeks of amenorrhea of three girls weighing from 1,950 to 2,300 g. Starting from a literature review of Kallmann-De Morsier syndrome, we discuss the role of LH during the follicular phase and the monitoring of ovarian stimulation.

Use of GNRH antagonists in reproductive medicine.

Gonadotrophin-releasing hormone (GnRH) plays a key role in the secretion of gonadotrophins, follicle-stimulating hormone (FSH) and luteinizing hormone (LH), which regulate steroidogenesis and folliculogenesis. Two GnRH antagonists, Cetrorelix and Ganirelix, deprived of histaminergic side-effects, have been introduced into ovarian stimulation protocols to prevent premature LH surges and proved their safety in clinical trials. At present, most of the published studies have not found significant differences in follicular recruitment, oocyte quality, and so on, except for a decrease in pregnancy and implantation rates in in vitro fertilization and embryo transfer (IVF-ET) cycles when the GnRH antagonist rather than the agonist was used. This decrease in pregnancy rates was in relation with a necessary learning curve of the physicians. Another possibility is the impact of the GnRH antagonist on endometrium through its GnRH receptor; this effect was cancelled after cryopreserved embryo transfers because the pregnancy rates were similar between GnRH antagonist and agonist in this case. GnRH antagonists were also interesting in poor responders and polycystic ovarian syndrome, where the agonists have not permitted to obtain the better results in IVF-ET cycles. Similarly, the GnRH antagonists could prevent the LH surge in the intrauterine insemination cycles.

Electroporation of immature and mature dendritic cells: implications for dendritic cell-based vaccines.

Until now, studies utilizing mRNA electroporation as a tool for the delivery of tumor antigens to human monocyte-derived dendritic cells (DC) have focused on DC electroporated in an immature state. Immature DC are considered to be specialized in antigen capture and processing, whereas mature DC present antigen and have an increased T-cell stimulatory capacity. Therefore, the consensus has been to electroporate DC before maturation. We show that the transfection efficiency of DC electroporated either before or after maturation was similarly high. Both immature and mature electroporated DC, matured in the presence of an inflammatory cytokine cocktail, expressed mature DC surface markers and preserved their capacity to secrete cytokines and chemokines upon CD40 ligation. In addition, both immature and mature DC can be efficiently cryopreserved before or after electroporation without deleterious effects on viability, phenotype or T-cell stimulatory capacity including in vitro antigen-specific T-cell activation. However, DC electroporated after maturation are more efficient in in vitro migration assays and at least as effective in antigen presentation as DC electroporated before maturation. These results are important for vaccination strategies where an optimal antigen presentation by DC after migration to the lymphoid organs is crucial.

MAGE-A genes are not expressed in human leukemias.

Immunotherapy is promising to improve the prognosis of human leukemias, at least as adjuvant treatment. Tumor-associated antigens such as antigens encoded by MAGE-A1, -A2, -A3, -A4, -A6 and -A12 genes might provide tools in this field. We demonstrated recently that the presentation peptides encoded by MAGE-A genes might make leukemic blasts suitable targets to cytolytic T lymphocytes. We reported previously negative data of MAGE-A1 gene expression in hematological malignancies, but in further studies positive results of MAGE-A gene expression were published in some subtypes of hematological malignancies such as T leukemia, myeloma and Hodgkin's disease. This led us to enlarge the screening of MAGE-A gene expression in human leukemias. In the RT-PCR screening of a large panel including 154 patients, only weak signal were detected in a few samples. We conclude that MAGE-A genes are not expressed in human leukemias.

An alternative open reading frame of the human macrophage colony-stimulating factor gene is independently translated and codes for an antigenic peptide of 14 amino acids recognized by tumor-infiltrating CD8 T lymphocytes.

We show that cytotoxic T lymphocytes (CTLs) infiltrating a kidney tumor recognize a peptide encoded by an alternative open reading frame (ORF) of the macrophage colony-stimulating factor (M-CSF) gene. Remarkably, this alternative ORF, which is translated in many tumors concurrently with the major ORF, is also translated in some tissues that do not produce M-CSF, such as liver and kidney. Such a dissociation of the translation of two overlapping ORFs from the same gene is unexpected. The antigenic peptide encoded by the alternative ORF is presented by human histocompatibility leukocyte antigen (HLA)-B*3501 and has a length of 14 residues. Peptide elution indicated that tumor cells naturally present this 14 mer, which is the longest peptide known to be recognized by CTLs. Binding studies of peptide analogues suggest that it binds by its two extremities and bulges out of the HLA groove to compensate for its length.

Expression of prostate-specific membrane antigen in transitional cell carcinoma of the bladder: prognostic value?

The expression of Prostate-specific membrane antigen (PSMA) mRNA was assessed in the normal bladder urothelium (n = 9), transitional cell carcinoma (TCC) specimens (n = 52), TCC-derived cell lines (n = 3), and preoperative blood samples from TCC patients (n = 27). Specific PSMA mRNA was found in 100% of normal and malignant tissues and two cell lines. PSMA protein was detected in normal (n = 3) and malignant tissues (n = 4). Using a PSMA-specific substrate, PSMA enzymatic activity was found in two bladder cell lines and correlated with immunostaining. Seven of the 27 TCC preoperative blood samples were positive by reverse transcription-PCR. These preliminary results, obtained on a nonrandomized cohort of patients, correlated with tumor invasion (positive RT-PCR: 0% for pT < or = 2 versus 41% for pT > or = 3) and 2-year survival rate (81% in the PSMA-negative group versus 29% in the PSMA-positive group). Although the clinical usefulness of this assay requires confirmation in larger prospective randomized trials, current preliminary results suggest that a blood-borne PSMA mRNA PCR assay may be a useful tool to predict a poor outcome in TCC patients.

Monoclonal antibody 57B stains tumor tissues that express gene MAGE-A4.

Monoclonal antibody (Mab) 57B, which was raised against a recombinant MAGE-A3 protein, was tested for its ability to stain cells expressing various members of the MAGE-A gene family. COS-7 cells transfected with cDNAs encoding MAGE-A1, A2, A3, A4, A6, or A12 were stained, whereas those transfected with MAGE-A8, A9, A10, or A11 cDNAs were not. However, in tissue sections, we observed a different pattern of staining: the antibody effectively stained the tumors that expressed MAGE-A4 and only these tumors, regardless of the expression of the other MAGE-A genes. It seems, therefore, that at the level of MAGE gene expression found in tumors, a level clearly lower than that observed in transfected COS cells, only the MAGE-A4 protein can be reliably detected. We conclude that the 57B Mab should be useful for tumor diagnosis related to therapeutic vaccination involving MAGE-A4.

Expression of gene MAGE-A4 in Reed-Sternberg cells.

Genes of the MAGE-A family are expressed in several types of solid tumors but are silent in normal tissues with the exception of male germline cells, which do not carry HLA molecules.Therefore, peptides encoded by MAGE-A genes are strictly tumor-specific antigens that can be recognized by CTL and constitute promising targets for immunotherapy. The expression of 6 genes of the MAGE-A family was tested with reverse transcriptase-polymerase chain reaction in lymphoma samples. Among 38 samples of non-Hodgkin lymphoma, 1 anaplastic large cell lymphoma expressed genes MAGE-A1, -A2, -A3, -A4, and -A12, and 1 lymphoepithelioid T-cell lymphoma expressed gene MAGE-A4. Five of 18 samples (28%) from patients with Hodgkin disease expressed gene MAGE-A4. In tissue sections, staining by a monoclonal antibody that recognizes the MAGE-A4 protein was observed in 11 of 53 samples (21%) from patients with Hodgkin disease. In the positive samples, the Reed-Sternberg cells were strongly stained whereas the surrounding cells were not. These results indicate that Hodgkin disease may be a target for specific immunotherapy involving MAGE-A4 antigens.

Cytolytic T lymphocytes raised against a human bladder carcinoma recognize an antigen encoded by gene MAGE-A12.

Human bladder carcinoma line LB831-BLC expresses several distinct Ags that are recognized by different autologous CTL. Here, we show that one of these Ags is presented by HLA-Cw7 and encoded by gene MAGE-A12. This is the first time that CTL directed against a MAGE-encoded Ag have been derived from the lymphocytes of a patient with cancer other than melanoma. This new Ag was found to be nonapeptide VRIGHLYIL, corresponding to position 170-178 of the MAGE-A12 protein. Gene MAGE-A12 is silent in normal tissues except in male germline cells, which do not express HLA molecules. It is expressed in 26-62% of melanomas, infiltrating bladder carcinomas, lung carcinomas, esophageal carcinomas, and head and neck carcinomas. Because HLA-Cw7 is present in 43% of Caucasians, this new Ag is shared by many tumors and should be a useful target for cancer immunotherapy.

Processing of some antigens by the standard proteasome but not by the immunoproteasome results in poor presentation by dendritic cells.

By stimulating human lymphocytes with an autologous renal carcinoma, we obtained CTL recognizing an antigen derived from a novel, ubiquitous protein. The CTL failed to lyse autologous EBV-transformed B cells, even though the latter express the protein. This is due to the presence in these cells of immunoproteasomes, which, unlike standard proteasomes, cannot produce the antigenic peptide. We show that dendritic cells also carry immunoproteasomes and fail to present this antigen. This may explain why the relevant CTL escape thymic deletion and are not regularly activated in the periphery. Lack of cleavage by the immunoproteasome was also observed for melanoma differentiation antigen Melan-A26-35/HLA-A2, currently used for antitumoral vaccination. For immunization with such antigens, proteins should be less suitable than peptides, which do not require proteasome digestion in dendritic cells.

Quantitative evaluation of the expression of MAGE genes in tumors by limiting dilution of cDNA libraries.

The MAGE-A genes are expressed in tumor cells but not in healthy tissues, except in male germ line cells and in placenta. They encode tumor-specific antigens recognized by autologous cytolytic T lymphocytes (CTLs). On the basis of semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) assays, 6 of the 12 members of the MAGE-A family, including MAGE-A1, were previously reported to have a high level of expression in tumors, whereas 5 other members, including MAGE-A10, were expressed at a much lower level, deemed to be insufficient for CTL recognition. However, analysis with antibodies has shown that some melanoma cell lines contain equivalent amounts of MAGE-A1 and MAGE-A10 proteins. This discrepancy appeared to be due to the low efficacy of the primers that had been used for the previous MAGE-A10 RT-PCR assays. This led us to develop a method that is independent of the efficacy of the PCR primers to evaluate MAGE-A gene expression. cDNA libraries from tumor cell lines were introduced into bacteria, of which 200 pools of about 500 bacteria were maintained in microcultures. The frequencies of the MAGE-A cDNA clones in each library were evaluated by performing PCR assays on each of these pools. The abundance of MAGE-A10 cDNAs was found to be similar to that of MAGE-A1 in 3 of the libraries that were analyzed, including 2 with high expression (1/6,400), confirming that MAGE-A10 is expressed at a high level. MAGE-A2, A3, A4, A6 and A12 cDNAs were also confirmed often to be present at a frequency of more than 1/10,000, a level of expression that should suffice for recognition of antigenic peptides encoded by these genes by cytolytic T cells. The remaining MAGE genes are either not expressed in tumors or are expressed at a very low level, with the exception of MAGE-A8 and 11, which show high expression in a very small number of tumors. This method also allowed us to isolate 5 MAGE-A cDNAs that we had not obtained previously, enabling us to delineate the exons in the sequences of genes MAGE-A5, A8, A9, A10 and A11.

A new MAGE gene with ubiquitous expression does not code for known MAGE antigens recognized by T cells.

A number of genes of the MAGE family have been shown to code for antigens that are recognized on many human tumors by autologous CTLs. These antigens should be strictly tumor specific because the encoding MAGE genes are not expressed in normal adult cells, except for male germ-line cells, which lack HLA expression. Here, we report that a distant relative of the previously identified MAGE genes is expressed in many, if not all, normal tissues. This gene, which was named MAGE-D, is located in Xp11. Its exon-intron structure is completely different from that of the other MAGE genes. None of the 20 MAGE antigenic peptides presently known to be recognized by T lymphocytes is encoded by the new MAGE gene. It appears, therefore, that this new finding leaves intact the tumor specificity of the antigens encoded by the MAGE genes that are expressed only in tumor and germ-line cells.