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Paris le 20 septembre 2018Le témoignage qui suit est basé sur les récits de nos parents, Tauba Mandelbaum née Weissblum et Lejbus (Léon) Mandelbaum, concernant la journée du 11 septembre 1942. Cette journée s’inscrit dans notre histoire familiale comme une pierre à deux faces, l’une noire, l’autre blanche. Le 11 septembre 1942, jour de Rosh Hashana, nos parents sont réveillés, vers 5h30 du matin, par des coups frappés à la porte de leur maison, 6 rue du Marais à Lens. Un gendarme français et...

To the editor: The prognosis of sickle cell anemia (SCA), initially disastrous in the severe forms of the disease, has greatly improved, leading to increased life expectancy. More than 95% to 99% of the children diagnosed with SCA in developed countries will become young adults, reaching

Frozen thawed embryo transfer is currently an important part of present-day assisted reproductive technology (ART) aiming at increasing the clinical pregnancy rate per oocyte retrieval. Although slow freezing method has been the reference during 2 decades, the recent years witnessed an expansion of ultrarapid cryopreservation method named vitrification. Recently in France, vitrification has been authorized for cryopreserving human embryos. Therefore BLEFCO consortium decides to perform a descriptive study through questionnaires to evaluate the state of vitrification in the French clinical practice. Questionnaires were addressed to the 105 French centres of reproductive biology and 60 were fully completed. Data analysis revealed that embryo survival rate as well as, clinical pregnancy rate were increased after vitrification technology when compared to slow freezing procedure. Overall, these preliminary data suggest that vitrification may improve ART outcomes through an increasing of the cumulative pregnancy rate per oocyte retrieval.

Auteur(s) : Jacqueline Mandelbaum (membre du Conseil d’orientation de l’Agence de la biomedecine)* jacqueline.mandelbaum@tnn.aphp.fr * ancienne chef du service de Biologie de la reproduction/CECOS (Centre d’etude et de conservation des oeufs et du sperme) de l’hopital Tenon, APHP, Paris 6, 75020 Paris, France Voir version PDF.

Land-based investments can create significant grievances for local individuals or communities, and host governments seeking to address those grievances must navigate a complicated landscape of legal obligations and pragmatic considerations. This briefing note, funded by UK aid from the Department for International Development, focuses on practical solutions for governments confronting grievances that arise from large-scale investments in agricultural or forestry projects. It accompanies a more in depth report on similar issues, entitled Land deal dilemmas: Grievances, human rights, and investor protections. The briefing note considers such solutions in the context of governments’ legal obligations, particularly those imposed by international investment law, international human rights law, and investor-state contracts. Understanding the implications of this diverse range of legal obligations is particularly important in light of investors’ growing recourse to international investment arbitration, which can expose a government to liability under an international investment treaty for actions that may be in the best interest of a country and its citizens. Analyzing such obligations is a useful first step for a government seeking to protect its citizens against the negative impacts of land-based investments.

International audience; Study question: In couples who have chosen and confirmed the fate of surplus frozenembryos, which factors influence their decision, with a special emphasis on their symbolic representation of the embryo(s)? summary answer: Embryo representation and gamete donation use significantly influence the fate of surplus cryopreserved embryos. what is known already: Previous studies report difficulties for couples to decide whether or not to continue storing their frozen embryo(s) and different factors have been already highlighted which influence their decision, including embryo conceptualization, information and support provided by the medical institution, quality of embryo(s) and life events. Little is known, however, about couples who definitely decided to stop their parental project and finalized the process of decision-making about the fate of their cryopreserved embryo(s). study design, size, duration: This prospective study was conducted over a period of 3 years (2007-2010) and included IVF/ICSI patients with surplus frozen embryos, who made a final embryo disposition decision. Among the 280 eligible IVF/ICSI patients, 247 agreed to participate in the study. According to the available options, 91 persons chose to 'stop cryopreservation', 77 chose donation to 'research' and 48 'embryo donation' to infertile couples. Furthermore, 31 participants who chose embryo donation for a parental project were refused by the center as not compatible with their mandatory medical conditions. Among them, 27 participants then selected donation to research as a new option and were included in a fourth group: 'donation to research after Refusal of Embryo Donation for parental project' or 'research- RED' (n = 27). Four participants chose 'stop cryopreservation', however, given the small number of subjects this latter group was not included in the analysis. In all, 243 participants who made a final choice concerning the fate of their cryopreserved embryos were included in this study. participants/materials, setting, methods: Participants were sent a letter of invitation to a semi-structured interview of 30 min with a psychologist. Interviews were conducted separately for each partner, including a questionnaire with a common part and a specific part, according to the chosen option, and allowing a quantitative evaluation. A multivariate logistic regression model was used to assess the link between their embryo representation and their decision about their embryos' fate. main results and the role of chance: After adjustment for age, gender, gamete donation, number of children and the different embryo representations, a choice to 'stop cryopreservation' is more frequent if the embryo is represented as a child [odds ratio (OR) adjusted = 3.29, 95% confidence interval (CI) = 1.62'6.66], P = 0.0009. Representing the embryo as a project prompts patients to choose 'donation to research' [OR adjusted = 3.76, 95% CI = 1.56'9.06], P = 0.0032. Respondents are more likely to choose 'embryo donation' if they represent the embryo as a potential person [OR adjusted = 3.77, 95% CI = 1.45'9.80], P = 0.0064. Furthermore, patients who benefited from gamete donation are 10 times more likely to donate their embryos to another couple [OR adjusted = 10.62, 95% CI = 3.99'28.30], P , 0.0001. For more than half the participants (57%) the decision-making was easy, however, deciding to stop cryopreservation was significantly more difficult than choosing research or embryo donation (P , 0.0001). limitations, reasons for caution: Socio-economic status, moral and religious affiliations are known to influence the choice of couples but analyzing these factors was not an aim of the present study. wider implications of the findings: When couples definitely decide to stop their parental project, the embryo symbolic representation remains the main factor that influences the fate of their frozen embryo(s). Moreover, this representation can evolvewhen influenced by external events and information provided. In order to support patients who are making this difficult decision, it could be helpful to explore this symbolic representation early in the IVF/ICSI procedure, before surplus embryo freezing, as a new tool enhancing the accuracy of counseling. study funding/competing interest(s): this study was supported by a grant from the 'Agence de la biomedicine (ABM)', the national regulatory ART agency, under the authority of the French Ministry of Health. The authors have no conflict of interest to declare. © The Author 2016.

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. Hypogonadotrophic hypogonadism (HH) is characterized by deficient gonadotrophin secretion, resulting from pituitary or hypothalamic defects. In order to induce spermatogenensis, 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 a paternity. In order to evaluate the nuclear integrity of induced sperm cells, sperm samples from treated HH patients were compared to sperm samples from normospermic control donors. Sperm cells were analysed by FISH, using probes specific for chromosomes 13, 21, 18, X and Y, and by TUNEL 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.

DAX1/NR0B1 mutations are responsible for X-linked congenital adrenal hypoplasia (AHC) associated with hypogonadotropic hypogonadism (HH). Few data are available concerning testicular function and fertility in men with DAX1 mutations. Azoospermia as well as failure of gonadotrophin treatment have been reported. We induced spermatogenesis in a patient who has a DAX1 mutation (c.1210C>T), leading to a stop codon in position 404 (p.Gln404X). His endocrine testing revealed a low testosterone level at 1.2 nmol/l (N: 12-40) with low FSH and LH levels at 2.1 IU/l (N: 1-5 IU/l) and 0.1 IU/l (N: 1-4 IU/l), respectively. Baseline semen analysis revealed azoospermia. Menotropin (Menopur(®):150 IU, three times weekly) and human chorionic gonadotrophin (1500 IU, twice weekly) were used. After 20 months of treatment, as azoospermia persisted, bilateral multiple site testicular biopsies were performed. Histology revealed severe hypospermatogenesis. Rare spermatozoa were extracted from the right posterior fragment and ICSI was performed. Four embryos were obtained and, after a frozen-thawed single-embryo transfer, the patient's wife became pregnant and gave birth to a healthy boy. We report the first case of paternity after TESE-ICSI in a patient with DAX1 mutation, giving potential hope to these patients to father non-affected children. Furthermore, this case illustrates the fact that patients with X-linked AHC have a primary testicular defect in addition to HH.

Resume Objectif Au cours des fecondations in vitro (FIV) classiques, 5 % des tentatives se soldent par un echec de fecondation imprevisible, du fait d’une absence d’anomalies des parametres spermatiques. Dans 56 % des cas, aucune cause ovocytaire evidente n’est retrouvee. On observe, par contre, un defaut ou une absence de fixation des spermatozoides a la zone pellucide (ZP). Cette etude a pour objectif de preciser les causes moleculaires d’origine spermatique des echecs de FIV, qui sont a ce jour imprevisibles d’apres les donnees de la spermiologie classique. Patientes et methodes Le profil d’expression proteique des spermatozoides de sujets, presentant un echec complet de fecondation et un defaut de fixation des spermatozoides a la ZP, est compare a celui de temoins (hommes a sperme normal et fecondant, participant a une FIV d’indication tubaire). Pour cela, les echantillons de sperme sont analyses par la technologie d’electrophorese 2 Dimensional Electrophoresis-Differential In Gel Electrophoresis (2D-DIGE) avec au prealable une etape de pre-fractionnement des extraits proteiques selon leur point isoelectrique (fraction acide, intermediaire et basique). Resultats Sept proteines differentiellement exprimees chez tous les cas et chez tous les temoins ont ete mises en evidence dans la fraction acide et sept dans la fraction basique. Douze d’entre elles ont ete identifiees par spectrometrie de masse, dont deux (le recepteur a la laminine LR67 et la L-xylulose reductase) ont deja fait la preuve de leur implication dans l’interaction gametique. Discussion et conclusion Ces proteines meritent des investigations complementaires visant a determiner si certains echecs de fecondation en FIV sont causes par des anomalies au niveau de leur expression.

BACKGROUND: Conflicting results have been published about the determinants of pregnancy after oocyte donation (OD). We used the OD model to determine predictive factors of pregnancy in the recipient after frozen-thawed embryo transfer (FTET) in a specific series where all the embryos were cryopreserved without any prior selection for fresh transfer. METHODS: We report a retrospective study in a university tertiary care center. Multivariate analysis and logistic regression were used to identify predictive factors of pregnancy in a series of 450 OD FTET cycles in 198 infertile women between January 1992 and December 2006. RESULTS: The mean (± SD) recipient age was 35.7 (± 4.5). Impaired ovarian function was the main indication for OD. The mean ± SD (range) number of embryos transferred was 1.65 ± 0.5 (1-3). Overall clinical pregnancy, implantation and delivery rates were 30, 18 and 23%, respectively. After univariate analysis, pregnancy rates were significantly higher in recipients under 35 years, in women with a body mass index (BMI)

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.

The aim of this study is to determine if Müllerian agenesis has a genetic basis linked to the WNT genes. Genomic DNA analyses for mutations in the coding sequences of four members of this family in a series of 11 women with Mayer-Rokitansky-Kuster-Hauser syndrome found four variants in the coding sequence of these genes, but causal mutations were not observed. This supports the hypothesis that mutations in the coding sequence of WNT4, WNT5A, WNT7A, and WNT9B are not responsible for the Mayer-Rokitansky-Kuster-Hauser syndrome.

Le chromosome Y humain contient de nombreux genes et familles de genes necessaires a la spermatogenese. La majeure partie de ces genes est localisee au sein de sequences repetees frequemment soumises a des rearrangements. Les deletions des regions AZFa, AZFb et AZFc sont retrouvees chez 10 a 15 % des hommes presentant une oligozoospermie severe ou une azoospermie. Plusieurs deletions partielles de la region AZFc ont ete decrites. L'une d'entre elles emporte la moitie des genes de la region AZFc. Ses consequences sur la fertilite dans la population eurasienne semblent nulles ou minimes. Une autre deletion partielle denommee gr/gr emporte plusieurs genes de la region AZFc et a ete proposee comme un facteur de risque d'infertilite. Neanmoins, cette relation causale n'est pas evidente car il existe en fait plusieurs varietes de deletions gr/gr et leur repartition varie considerablement en fonction des origines ethniques et geographiques. Ces deletions semblent fixees sur certaines lignees de chromosome Y et n'auraient que peu ou pas d'influence sur la fertilite. La majorite des donnees concernant l'organisation chromosomique et le contenu genique du chromosome Y a ete deduite de la sequence de reference du chromosome Y deposee dans la base de donnees du NCBI. L'etablissement de donnees sur ce type de rearrangement dans la population generale n'a ete que recemment mis en œuvre. Ces etudes recentes ont souligne l'extreme diversite des polymorphismes de structure du chromosome Y dans la population generale. Correler ces polymorphismes de structure a la variabilite phenotypique sera l'enjeu de futures etudes. Il est vraisemblable que ces etudes aboutiront a la caracterisation non plus d'une sequence de reference unique du chromosome Y mais de plusieurs sequences de reference.

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.

Recently, mutations in the X-linked ubiquitin protease 26 (USP26) gene have been proposed to be associated with male infertility. In particular a 371insACA, 494T>C and 1423C>T haplotype, which results in a T123-124ins, L165S and H475Y amino acid change respectively, has been reported to be associated with Sertoli cell-only syndrome (SCOS) and an absence of sperm in the ejaculate. Here, we demonstrate that two of these changes actually correspond to the ancestral sequence of the gene and that the USP26 haplotype is present in significant frequencies in sub-Saharan African and South and East Asian populations, including in individuals with known fertility. This indicates that the allele is not associated with infertility. The pattern of frequency distribution of the derived haplotype (371delACA, 494T), which is present at high frequencies in most non-African populations could be interpreted as either a result of migration followed by simple genetic drift or alternatively as positive selection acting on the derived alleles. The latter hypothesis seems likely, because there is evidence of strong positive selection acting on the USP26 gene.

a Service de gynecologie–obstetrique et medecine de la reproduction, hopital Tenon, 4, rue de la Chine, 75970 Paris cedex 20, France b Cabinet Audebert et Parneix, 35, rue de Turenne, 33000 Bordeaux, France c Centre d’AMP, clinique Saint-Antoine, 696, rue Robert-Pinchon, 76230 Paris Guillaume, France d Service de gynecologie–obstetrique, CHI Jean-Rostand, 141, Grande-Rue, 92311 Sevres, France e Inserm U 149, 16, avenue Paul-Vaillant-Couturier, 94807 Villejuif cedex, France f Inserm U 149, 123, boulevard de Port-Royal, 75014 Paris, France g Centre de sterilite, 8, rue de Marignan, 75008 Paris, France h Maternite–AMP, hopital Saint-Vincent-de-Paul, 82, avenue Denfert-Rochereau, 75014 Paris cedex 14, France i Service de gynecologie–obstetrique, hopital Antoine-Beclere, 157, rue de la Porte-de-Trivaux, 92140 Clamart, France j 15, rue Labrouste, 75015 Paris, France k Unite de medecine de la reproduction, CHUV, 1005 Lausanne, Suisse l 15, rue Faraday, 75017 Paris, France m Service de gynecologie–obstetrique, hopital Jean-Verdier, avenue du 14-Juillet, 93143 Bondy cedex, France n Service d’histologie, biologie de la reproduction et cytogenetique, hopital Tenon, 4, rue de la Chine, 75970 Paris cedex 20, France o Laboratoire de fecondation in vitro, clinique Saint-Jean-de-Languedoc, 20, route de Revel, 31400 Toulouse, France p Unite de recherche U 292, hopital de Bicetre, 82, rue du General-Leclerc, 94276 Le Kremlin-Bicetre, France q Service de gynecologie–obstetrique et de medecine de la reproduction, 123, boulevard de Port-Royal, 75014 Paris, France r Departement de gynecologie obstetricale et reproduction humaine, biologie de la reproduction, CHRU Bretonneau, 37044 Tours cedex, France

International audience; Objective: To identify the male molecular causes of failures of IVF (with a deficient binding of spermatozoa to the zona pellucida, without any obvious oocyte anomaly), which are undetected by classical sperm analysis. Design: Case-control prospective study. Setting: University hospital. Patient(s): Proteomic profiles of spermatozoa in patients with a complete failure of fertilization and no spermatozoa bound to the zona pellucida were compared with those of controls (men with normal fertilization and cleavage rates after classical IVF for tubal indication). Intervention(s): All samples were analyzed by two-dimensional fluorescence difference gel electrophoresis (2D-DIGE) after being divided into three fractions according to their isoelectric point. Main Outcome Measure(s): Differentially expressed proteins between infertile men and controls were identified by mass spectrometry. Result(s): Seventeen proteins differentially expressed between cases and controls were found. Twelve of these proteins were identified by mass spectrometry, and two may influence gametes interaction: laminin receptor LR67 and L-xylulose reductase (P34H). Conclusion(s): This study shows that 2D-DIGE might be useful in finding potential targets for diagnosis and prognosis of idiopathic infertility in IVF.

RESUME Il s’agit de l’exemple type des problemes poses par la medecine moderne. Nous avons mene a l’Hopital Tenon a Paris, entre 1994 et 1999, une etude retrospective interessant 177 transferts d’embryons congeles puis decongeles chez 139 patientes parmi les 300 demandeuses d’un don d’ovocytes pendant cette meme epoque. Ceci nous conduit a bien souligner les difficultes ethiques creees par la promulgation des lois de bioethique de 1994. C’est ainsi que deux consequences sont devenues evidentes : la penurie de plus en plus grande de donneuses et la necessite de ne transferer que des embryons congeles/decongeles due au decret de 1996 sur la Securite Sanitaire, qui impose une quarantaine des dits embryons pendant 6 mois. En contrepartie, l’emploi de cette methode a apporte de nouvelles informations sur l’implantation et souligne l’importance de la qualite de l’embryon qui est tres etroitement correlee a l’âge de la donneuse.

Human embryo cryopreservation represents an indispensable extension of in-vitro fertilization (IVF) programmes as long as they are based upon the recovery of a large number of oocytes. The most widely used procedures include the cryopreservation of human zygotes or embryos in early cleavage, using 1,2-propanediol and sucrose as cryoprotectants. Our results over a 10 year period (1986-1995) on 5032 thawed cycles involving 14 222 stored embryos make it possible to appraise the results and the contribution of embryo freezing to assisted reproduction. Embryos survived the freeze-thaw process in 73% of cases leading to 4590 transfers of 2.2 embryos (91% of thawed cycles). The clinical pregnancy rate per transfer was 16%, the live birth rate 12%, and the rate of babies born alive per transferred embryo was 6%. Embryo freezing monitored 10 years later produced an average of 8% of additional births. By then, 86% of stored embryos had been thawed for transfer to patients. Destruction or donation were required for only 8% of all frozen embryos and there was no news from the parental couple in relation to almost 6% of embryos. The fate of the vast majority of embryos was decided during the first 5 years of storage. Blastocyst cryopreservation is making new strides, thanks to co-culture systems and embryo selection. Micromanipulation procedures seem to have little impact on the outcome of embryo freezing. Human oocyte freezing is again clinically applied. Indeed, much of the concern about injuries to the oocyte structures through the freeze-thaw process do not seem to be justified, and the problems with frozen-thawed oocyte fertilization has been overcome using intracytoplasmic sperm injection (ICSI). As long as oocyte in-vitro maturation is not well controlled, better results will probably be obtained with mature oocyte cryopreservation. Emerging methods include the freezing of immature oocytes, follicles and ovarian tissue.

Objective. The objective of this study was the determination of sex chromosome aneuploidy frequency in spermatozoa from patients included in an in vitro fertilization (IVF) or intra cytoplasmic sperm injection (ICSI) protocol. Methods. Spermatozoa from nineteen patients, including patients with normal seminal parameters according to World Health Organization (WHO) criteria and patients exhibiting abnormal seminal parameters, were analyzed by dual color fluorescence in situ hybridization (FISH) for aneuploidies of the X and Y chromosomes. Our technique, using only probes for sex chromosomes and not for autosomes, does not discriminate between hyperhaploid and diploid sperm nuclei. The results were analyzed in two different ways: in relation to the semen status, denoted normal or abnormal and with regard to the ability of the sperm to fertilize oocytes when IVF or ICSI was performed. Results. Abnormal semen showed a significant increase in the overall rate of sperm nuclei with XY, XX and YY sex chromosome complements, 1.59% compared to normal semen, 0.78% (p

Angiotensin converting enzyme (ACE) is a metalloprotease with two isoforms. The somatic isoform is a key component of the renin-angiotensin system; its main function is to hydrolyse angiotensin I into angiotensin II. The germinal or testicular isoform (tACE) located at the plasma membrane of the spermatozoa, plays a crucial role in the spermatozoa-oocyte interaction during in vivo fertilization, in rodents. Disruption of the tACE in mice has revealed that homozygous male tACE-/- sire few pups despite mating normally. Few spermatozoa from these tACE-/- mice are bound to the zona pellucida (ZP) despite normal semen parameters. Based on these findings in mice models, we hypothesized that some infertile men that have the same phenotype as the tACE-/- mice, ie normal semen parameters and a lack of sperm bind to the ZP in vitro, may have a tACE defect.Twenty four men participated to this study. The case subjects (n = 10) had normal semen parameters according to the WHO guidelines (WHO 1999) but a total in vitro fertilization failure with absence of sperm fixation to the ZP. The control subjects (n = 14) also had normal semen parameters and a normal fertilization rate ≥65%. We investigated the tACE expression in spermatozoa by Western-Blot and performed a DNA sequencing of the tACE gene.Three case-subjects and one control-subject had no tACE expression. There were no statistic differences between the two groups. No mutation was detected in the tACE DNA sequence.Our results didn't show any involvement of tACE in human fertilization especially in ZP binding.L’enzyme de conversion de l′angiotensine (ACE) est une métalloprotéase qui se présente sous deux isoformes. Une isoforme somatique qui intervient dans la régulation de la pression artérielle via le système rénine-angiotensine et une isoforme testiculaire spermatique (ACEt) qui interviendrait dans la fixation du spermatozoïde aux enveloppes ovocytaires (zone pellucide et/ou oelemme). Des expériences d’invalidation du gène de l′ACEt chez la souris ont montré que les mâles homozygotes ACEt−/− présentaient une faible descendanceVingt quatre patients ont été inclus dont 10 cas avec un sperme normal et un échec total de fécondation lors d’une FIV et 14 témoins ayant les mêmes caractéristiques spermatiques mais présentant de bons taux de fécondation (≥65%). L’étude de l’expression protéique de l’ACEt a été faite par western-blot sur les culots de spermatozoïdes. Un séquençage du gène codant pour l′ACEt a été fait sur l′ADN lymphocytaire.Trois cas et un témoin présentaient un défaut d’expression de l′ACEt sans diffèrence statistiquement significative entre ces deux groupes. En PCR, aucune mutation n’a été retrouvée après séquençage du gène de l′ACEt.Nos résultats n’ont pas objectivé une implication de l′ACEt dans l′interaction entre ovocyte et spermatozoïde et notamment dans la fixation à la ZP.

The expression of both components of the high-affinity leukaemia inhibitory factor receptor, LIFR beta and glycoprotein 130 (gp130), was investigated in human oocytes and individual in-vitro cultured preimplantation embryos by reverse transcription-polymerase chain reaction (RT-PCR). Messenger RNA of both LIFR beta and gp130 was detected in as little as 1/30 and 1/12 sample equivalents of cDNA respectively, in oocytes (n = 4), 4-cell and expanded, blastacyst stage embryos. LIFR beta but not gp130 transcripts were detected at the 2-, 8- and 10-cell stages, and in cavitating and hatched blastocysts. In order to exclude a simian origin of these PCR products resulting from the Vero cell line that was used as a feeder during culture to the blastocyst stage, they were digested with restriction endonucleases Taql (LIFR beta) or Kpnl (gp130). Their human origin was confirmed. The results support an earlier finding of LIFR beta mRNA expression in human blastocysts, and extend these results to earlier stages and oocytes. This is the first report of LIFR beta and gp130 transcription in human oocytes. Taken together these results demonstrate that transcription of LIFR beta and gp130 takes place throughout human preimplantation development, and suggest that functional LIF receptors might be present at these stages. These results further confirm the feasibility of performing mRNA phenotyping of multiple genes with RNA derived from a single preimplantation stage embryo.

A study, in which 110 patients were screened by a psychoanalyst, included 69 recipients who chose non-anonymous oocyte donation, i.e. they received oocytes from a known donor, most frequently a sister or a close relative. Another 41 recipients received anonymous oocytes, but had to bring a donor. Psychological motivations for either choice are reported, and significant topics such as attitudes towards confidentiality and links to the child are compared. No specific psychopathology is reported at this stage. An additional study on children born by these techniques is ongoing.

Of 44 pregnancies obtained at the Tenon Maternity Hospital by in vitro fertilization (IVF) using donated oocytes, 25 have already been completed by spontaneous delivery or cesarean section: 22 (88%) single; 2 (8%) twins, and 1 (4%) triple. The most frequently seen complications of pregnancy were hypertension (16%) and preterm labor in the last 3 months (12%). However, as compared to a control group of 275 pregnancies resulting from classical IVF, the only difference was a very high percentage of cesarean sections (72%). No fetal deaths or neonatal malformations were reported.

Objective To determine if there is a relationship between various forms of partial AZFc deletions and spermatogenic failure. Design Case-control study. Setting Infertility clinic (Tenon Hospital, Paris). Patient(s) 557 men, comprising 364 infertile men from mixed ethnic backgrounds, and 193 men with known fertility (n = 84) and/or normospermic (n = 109). Intervention(s) Characterization of 32 partial AZFc deletions. Main Outcome Measure(s) DAZ gene cluster divided into two families ( DAZ1/2 and DAZ3/4 ), CDY1 gene, and Y-chromosome haplogroups. Result(s) We observed 18 partial AZFc deletions in 364 (4.95%) infertile men compared with 14 out of 193 (7.25%) in the control normospermic/fertile group. Conclusion(s) The analysis of informative Y-chromosome single nucleotide variants combined with Y-chromosome haplogroup definition enabled us to infer seven deletion classes that occur on a minimum of six Y-chromosome parental architectures. We found no relationship between either the presence or the absence of DAZ1/2, DAZ3/4, CDY1a , or CDY1b with spermatogenic failure at least on one Y-chromosome lineage. The DAZ dosage and Southern blot analyses indicated that the majority of individuals tested carried two copies of the DAZ gene, indicating a partial AZFc deletion. Our data are consistent with the hypothesis that, at least in our study populations, partial AZFc deletions may have a limited impact on fertility.

Objective To raise the possibility that pregnancy can be obtained by assisted reproductive techniques in patients with human seminal plasma allergy. Design Case report. Setting University hospital. Patient(s) A woman consulted for a 3-year primary infertility. She reported lack of intercourse because of a seminal plasma allergy. Intervention(s) One intrauterine insemination associated with antihistamine treatment was performed with carefully washed spermatozoa. Result(s) Immediately after the insemination, the patient had an allergic reaction treated by steroids. Fortunately, this single attempt led to a successful pregnancy, and the patient gave birth to a healthy girl. Conclusion(s) Pregnancies can be obtained in patients with seminal plasma hypersensitivity by means of intrauterine insemination. However, serious complications may occur after performing intrauterine insemination, which is not a totally reliable method to prevent an allergic reaction. Thus a multidisciplinary team should follow such patients carefully and watch for the potential risks and side effects. In vitro fertilization may represent the more cautious option.

This is a report of a 6-year follow-up of a male patient's semen parameters during heavy chronic alcohol intoxication and after withdrawal. A slowly progressive negative impact of alcohol could be observed: isolated moderate teratozoospermia was firstly noted followed by oligoasthenoteratospermia. Then a severe worsening resulted in cryptozoospermia and ultimately in azoospermia. At this moment, the histological analysis of a testicular biopsy revealed a maturation arrest of the germinal cells at the pachytene stage with no mature sperm cells. Alcohol withdrawal was then obtained, allowing a very fast and drastic improvement of semen characteristics; strictly normal semen parameters were observed after no more than 3 months. Taking into consideration these data, patients should be questioned about their alcohol intake before assisted reproductive technology and should be informed about this adverse effect. Moreover, this case report emphasizes how quickly benefits can be obtained after withdrawal, even in the case of heavy chronic alcohol intake.

BACKGROUND: The metabolic pathway of folate is thought to influence DNA stability either by inducing single/double stranded breaks or by producing low levels of S-adenosyl-methionine leading to abnormal gene expression and chromosome segregation. Polymorphisms in the genes encoding enzymes in the folate metabolism pathway show distinct geographic and/or ethnic variations and in some cases have been linked to disease. Notably, the gene Methylenetetrahydrofolate reductase (MTHFR) in which the homozygous (TT) state of the polymorphism c.665C>T (p.A222V) is associated with reduced specific activity and increased thermolability of the enzyme causing mild hyperhomocysteinemia. Recently several studies have suggested that men carrying this polymorphism may be at increased risk to develop infertility. METHODOLOGY/PRINCIPAL FINDINGS: We have tested this hypothesis in a case/control study of ethnic French individuals. We examined the incidence of polymorphisms in the genes MTHFR (R68Q, A222V and E429A), Methionine synthase reductase MTRR; (I22M and S175L) and Cystathionine beta-synthase (CBS; G307S). The case population consisted of DNA samples from men with unexplained azoospermia (n = 70) or oligozoospermia (n = 182) and the control population consisted of normospermic and fertile men (n = 114). We found no evidence of an association between the incidence of any of these variants and reduced sperm counts. In addition haplotype analysis did not reveal differences between the case and control populations. CONCLUSIONS/SIGNIFICANCE: We could find no evidence for an association between reduced sperm counts and polymorphisms in enzymes involved in folate metabolism in the French population.

The human Y chromosome contains a number of genes and gene families that are essential for germ cell development and maintenance. Many of these genes are located in highly repetitive elements that are subject to rearrangements. Deletion of azoospermia factor (AZF) regions AZFa, AZFb, and AZFc are found in approximately 10-15% of men with severe forms of spermatogenic failure. Several partial AZFc deletions have been described. One of these, which removes around half of all the genes within the AZFc region, appears to be present as an inconsequential polymorphism in populations of northern Eurasia. A second deletion, termed gr/gr, also results in the absence of several AZFc genes and it may be a genetic risk factor for spermatogenic failure. However, the link between these partial deletions and fertility is unclear. The gr/gr deletion is not a single deletion but a combination of deletions that vary in size and complexity and result in the absence of different genes. There are also regional or ethnic differences in the frequency of gr/gr deletions. In some Y-chromosome lineages, these deletions appear to be fixed and may have little influence on spermatogenesis. Most of these data (gene content and Y chromosome structure) have been deduced from the reference Y chromosome sequence deposited in NCBI. However, recently there have been attempts to define these types of structural rearrangements in the general population. These have highlighted the considerable degree of structural diversity that exist. Trying to correlate these changes with the phenotypic variability is a major challenge and it is likely that there will not be a single reference (or normal) Y chromosome sequence but many.

Background Recent progress in the treatment of sickle cell disease, in particular the use of hydroxyurea, has considerably modified the prognosis of this disease. Many more patients now reach reproductive age. The objective of this study was to assess the potential impact of hydroxyurea on the semen of patients. Design and Methods In this retrospective multicenter study, we evaluated the sperm parameters and fertility of 44 patients and analyzed the potential impact of hydroxyurea. Results We report data from the largest series so far of semen analyses in patients with sickle cell disease: 108 samples were analyzed, of which 76 were collected before treatment. We found that at least one sperm parameter was abnormal in 91% of the patients before treatment, in agreement with published literature. All sperm parameters seemed to be affected in semen samples collected during hydroxyurea treatment, and this impairment occurred in less than 6 months, later reaching a plateau. Furthermore, after hydroxyurea cessation, while global results in 30 patients were not statistically different before and after hydroxyurea treatment, in four individuals follow-up sperm parameters did not seem to recover quickly and the total number of spermatozoa per ejaculate fell below the normal range in about half the cases. Conclusions The observed alterations of semen parameters due to sickle cell disease seem to be exacerbated by hydroxyurea treatment. Until prospective studies reveal reassuring findings, we suggest that a pre-treatment sperm analysis be performed and sperm cryopreservation be offered to patients before hydroxyurea treatment.

The human Y chromosome contains a number of genes and gene families that are necessary for spermatogenesis. Many of these genes are embedded in repetitive elements that are subject to deletion events. Deletions of azoospermia factor (AZF) regions AZFa, AZFb, and AZFc are found in approx 10–15% of men with either unexplained severe oligozoospermia or azoospermia. These deletions fall on different Y chromosome backgrounds and there is no evidence for a link between a Y-chromosome lineage and the presence or absence of an AZF deletion. Several partial AZFc deletions have been described. One of these, which removes around half of all the genes within the AZFc region, appears to be present as in inconsequential polymorphism in populations of northern Eurasia. A second deletion, termed gr/gr, results in the absence of several AZFc genes and has been suggested to be a genetic risk factor for spermatogenic failure. However, the link between the gr/gr deletion and infertility is more complex. First, the gr/gr deletion is actually not a single type of deletion but a combination of deletions that vary in size and complexity and result in the absence of different members of the deleted azoospermia (DAZ) gene family as well as other AZFc genes, such as CDY1. Second, there are regional or ethnic differences in the frequency of gr/gr deletions. In some Y-chromosome lineages, these deletion appear to be fixed and may have little influence on spermatogenesis. Third, these observations have influenced a number of association studies aimed to determine the relationship between the gr/gr deletion and male infertility. Consequently, some studies suggest that the gr/gr deletion confers a strong genetic susceptibility to reduced sperm counts, whereas others suggest that the genetic susceptibility may not exist or be limited to specific Y-chromosome haplotypes.

No phenotypic effect is observed in most inversion heterozygotes. However, reproductive risks may occur in the form of infertility, spontaneous abortions or chromosomally unbalanced children as a consequence of meiotic recombination between inverted and non-inverted chromosomes. An odd number of crossovers within the inverted segment results in gametes bearing recombinant chromosomes with a duplication of the region outside of the inversion segment of one arm and a deletion of the terminal segment of the other arm [dup(p)/del(q) and del(p)/dup(q)]. Using fluorescence in-situ hybridization (FISH), the chromosome segregation of a pericentric inversion of chromosome 1 was studied in spermatozoa of a inv(1)(p22q42) heterozygous carrier. Three-colour FISH was performed on sperm samples using a probe mixture consisting of chromosome 1p telomere-specific probe, chromosome 1q telomere-specific probe and chromosome 18 centromere-specific alpha satellite DNA probe. The frequency of the non-recombinant product was 80.1%. The frequencies of the two types of recombinants carrying a duplication of the short arm and a deletion of the long arm, and vice versa, were respectively 7.6 and 7.2%, and these frequencies were not statistically significant from the expected ratio of 1:1. Sperm-FISH allows the further understanding of segregation patterns and their effect on reproductive failure and allows an accurate genetic counselling.

Objective To raise the possibility that a familial chromosomal translocation associated with teratozoospermia can disrupt a gene necessary for flagellum assembly. Design Case report. Setting University hospital. Patient(s) Two brothers with infertility related to anomalies of meiotic division and of the flagella assembly, presenting the same balanced 5-12 autosomal translocation. Intervention(s) Several intracytoplasmic sperm injection (ICSI) cycles in our IVF department for both couples. Main Outcome Measure(s) Sperm analysis, karyotypes, electron microscopy, and fluorescence in situ hybridization (FISH) analysis of spermatozoa performed using probes coding for chromosomes X, Y, 13, 18, and 21. Result(s) In both brothers, sperm analysis indicated a tail stump syndrome. Electron microscopy analysis displayed complex abnormalities, which were probably related to meiotic errors. The FISH analysis indicated an increase of diploid germ cells. Karyotypes of both patients revealed the same balanced chromosomal t(5;12) (p15.1; q21) translocation. Results of ICSI cycles were comparable for both couples. A twin pregnancy was achieved in one of these two couples, but a spontaneous miscarriage occurred at 10 weeks of gestation. Conclusion(s) The flagella anomalies raise the possibility that the translocation disrupts a gene necessary for the flagellum assembly, although a mutation in a gene unrelated to the chromosome breakpoints cannot be excluded.

No statistically significant difference in deletion frequency was found between infertile patients and a control group. It is suggested that in some populations the gr/gr deletion might be an inconsequential polymorphism.

BACKGROUND: Assisted hatching (AH) techniques, designed for facilitating the embryo escape out of the zona pellucida (ZP) have been used in IVF centres since 1992. The initial indications for AH were patient's age, ZP thickness, high basal FSH and repeated IVF failures. Several retrospective and prospective studies assessing AH in these indications have given disparate results. Our aims were to evaluate the benefits of AH and immunosuppressive/antibiotic treatment (IA) in patients with either a poor prognosis of success, previous implantation failures or transfers of cryopreserved embryos. METHODS: Four IVF centres allocated 426 patients, randomized for AH and IA, into four groups of AH indications between 1997 and 1999. AH was performed with a diode laser. ZP thickness, opening size and embryo score were recorded. Outcome measures were implantation and delivery rates. RESULTS: Patients coming for a first or third transfer of cryopreserved embryos and poor prognosis patients admitted for a first trial did not benefit from AH. Even patients with repeated implantation failures of fresh embryos did not gain significantly from AH. CONCLUSIONS: Among AH indications, absence of implantation after several transfers of good quality embryos remains the strongest patient selection criterion. Prescription of an immunosuppressive/antibiotic treatment is essential.

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.

We report a case of monozygotic triplet pregnancy after the transfer of three frozen-thawed embryos. The outcome of the pregnancy was the birth of three healthy normal boys. The pregnancy was a monochorionic triamniotic triplet pregnancy. This is the first such case of triplets, although it seems that zygote splitting is significantly more common than normal in patients undergoing in-vitro fertilization.

To the Editor: “Parental imprinting” refers to an epigenetic marking of genes that results in monoallelic expression. This phenomenon plays a critical role in embryogenesis and development. The epigenetic modification of the genome involves methylation changes and the remodeling of chromatin-associated proteins (Li 2002). Imprints are established during the development of germ cells, and the reprogramming of imprinting occurs within the first days after fertilization (Reik and Walter 2001). The alteration of normal imprinting patterns is implicated in a number of human genetic diseases. Among them, the Beckwith-Wiedemann syndrome (BWS [MIM 130650]) is an overgrowth syndrome secondary to the dysregulation of the imprinted 11p15 region (Maher and Reik 2000). Numerous mechanisms are involved in BWS, and ∼70% of cases of BWS are related to epigenetic abnormalities at the 11p15 locus, mostly demethylation of the KvDMR1 region of the KCNQ1OT (previously called “LIT1”) gene (MIM 604115) (Engel et al. 2000; Bliek et al. 2001; Gaston et al. 2001; Weksberg et al. 2001; DeBaun et al. 2002). KCNQ1OT encodes a noncoding antisense transcript within intron 10 of the KCNQ1 gene (MIM 192500) (Lee et al. 1999; Mitsuya et al. 1999; Smilinich et al. 1999) and might be involved in the regulation of parental imprinting of the centromeric domain of the 11p15 region (Fitzpatrick et al. 2002). In sheep and cattle, epigenetic abnormalities have been shown to be involved in large offspring syndrome (LOS) (Young et al. 1998). Affected animals exhibit various phenotypes, including large size at birth. In both species, the syndrome is caused by the in vitro exposure of embryos, between fertilization and the blastocyst stage, to various unusual environments. LOS is related to the loss of imprinting of the IGF2 receptor gene (MIM 147280), which ensures internalization and degradation of IGF2 and displays an antiproliferative function (Young et al. 2001). In vitro preimplantation procedures in mice are also responsible for overgrowth, owing to the abnormal expression of various imprinted genes, particularly the genes located at distal chromosome 7 (h19 [MIM 103280] and igf2 [MIM 147470] genes), orthologous to the human 11p15 region (Humpherys et al. 2001; Rideout et al. 2001). In humans, a case of BWS was recently described after in vitro fertilization (IVF) (Olivennes et al. 2001). Moreover, two recent papers (DeBaun et al. 2003; Maher et al. 2003) described an increase in prevalence of assisted reproductive technologies (ART) in patients with BWS. De Baun et al. (2003) reported a sixfold increase (4.6% vs. 0.76% in the general population) and showed that four of the six patients for whom DNA was available exhibited an isolated demethylation of KvDMR1 in the KCNQ1OT gene. Maher et al. (2003) reported a threefold increase (4% vs. 0.997% in the general population) and demonstrated that two of the six patients on whom molecular analysis could be done also exhibited an isolated demethylation of KvDMR1. Our department is a reference center in France for molecular diagnosis of BWS, and patients are referred from various medical departments (neonatology, pediatrics, genetics, and fetopathology). We studied a series of 149 patients referred for overgrowth syndromes and diagnosed as BWS, since all of them exhibited genetic or epigenetic defects at the 11p15 locus. According to the inclusion criteria described elsewhere (Gaston et al. 2001), 102 patients exhibited a complete form of BWS, and 47 exhibited an incomplete form of BWS. The techniques used to analyze the 11p15 region have been described elsewhere (Gaston et al. 2000, 2001). Epigenetic changes concerned 104 (70%) patients, most of whom (n=90) exhibited a loss of KvDMR1 methylation. Fourteen patients (9.4%) exhibited isolated hypermethylation of the H19 gene. Forty-two patients exhibited a genetic defect: 11p15 uniparental disomy (n=35; 23.5%) and germline CDKN1C (MIM 600856) mutation (n=7; 4.7%). Three patients (2%) had a chromosomal abnormality. Six of the 149 patients were born following ART. Of note, these six patients exhibited the same epigenetic abnormality (isolated demethylation of KvDMR1 with a demethylation index varying 72%–100%) (fig. 1). All of them were sporadic cases, and one was a DZ twin. The clinical features of these patients and the procedures of ART used for their conception are summarized in table 1. As shown in table 1, the phenotypes of patients born after ART were not different from phenotypes of the other patients (n=84) with isolated demethylation of KVDMR1, with the exception that only one patient born after ART exhibited ear abnormalities. These children were issued from various ART procedures: classical IVF, intracytoplasmic sperm injection (ICSI), embryo freezing, and transfer on day 2, day 3, and day 5. More recent procedures, like ICSI (two of six patients) or blastocyst transfer (one of six patients), did not prevail over other techniques. The representation of ART (4%) in our series is three times higher than that in the general population (1.3%), according to the national report of the French Ministry of Health (9,930 of 770,000 live births resulting in 1,999 from IVF, ICSI, or frozen embryo transfer). On the basis of this report, we would have expected 1.94 of the 149 patients with BWS to be born as a result of ART. To test the significance of this difference of frequencies, we used the Fisher’s exact test (P=.01) as well as the Poisson approximation (two-tailed P=.018; 95% CI 1.5–8.7). Strength of the association between exposure to ART and risk of BWS is expressed by an odds ratio of 3.2 (95% CI 1.4–7.3). This rate is the same as that described by Maher et al. (2003) but lower than that described by DeBaun et al. (2003), which addressed a prospective study. In our series and in Maher’s series, this rate is probably underestimated, as specific questions regarding ART have only been asked systematically in the past year. Figure 1 Methylation analysis of KvDMR1 in liver tissue (patient 131) and leukocytes (patients 15, 94, 98, 115, and 137) from the six patients with BWS born after ART and in leukocytes from a normal control (C). Genomic DNA was digested with BamHI and the methylation-sensitive ... Table 1 Clinical Characteristics of the Six Patients with BWS Born Following ART Although the analysis of the imprinting status at chromosome 15q11-13 in children born after ICSI did not reveal an imprinting defect (Manning et al. 2000), two recent papers reported three patients with Angelman syndrome (MIM 105830) born after ICSI (Cox et al. 2002; Orstavik et al. 2002). All three patients exhibited an imprinting defect, which is a rare cause of Angelman syndrome. As in the previous two reports (DeBaun et al. 2003; Maher et al. 2003), our data suggest that ART may favor imprinting alterations at the centromeric imprinted 11p15 locus and, consequently, the incidence of BWS. These data highlight the need to carefully follow up children born after ART to test for BWS and other diseases related to imprinted regions. Although no specific procedures of ART appear to be associated with a risk of BWS in our series, these data lend support to the importance of precisely recording these different procedures of ART, particularly the stimulation protocol, the biological technique, the stage of maturation of the gametes, the culture media used at each step, and the timing of embryo transfer.

Objective To determine the impact of circulating LH concentrations during controlled ovarian hyperstimulation on the outcome of IVF. Design Retrospective study. Setting University hospital. Patient(s) Two-hundred seventy women who had a short stimulation protocol with GnRH antagonist and ovarian stimulation with recombinant FSH (rFSH). Intervention(s) GnRH antagonist and rFSH were administered SC; blood samples were collected on the day of GnRH antagonist administration, 1 day after, and on the day of hCG administration. Main outcome measure(s) A threshold of 0.5 IU/L on the day of hCG was chosen to discriminate between women with LH concentrations ≤0.5 IU/L (group A, n=119) or >0.5 IU/L (group B, n=151). Result(s) The two groups were comparable with regard to the clinical parameters. In group A, significantly lower LH concentrations were observed on day 9 of the cycle and on the day of hCG administration. The numbers of oocytes retrieved, embryos obtained, and embryos cryopreserved were significantly higher in group A compared with group B. The proportion of clinical pregnancies was similar in the two groups (21.1% vs. 22.7 % per ET). Conclusion(s) In GnRH antagonist and rFSH protocols, suppressed serum LH concentrations do not have any influence on the final stages of follicular maturation, pregnancy rates, or outcomes.

Education has always been a priority for the European Society of Human Reproduction and Embryology (ESHRE). Many efforts have been dedicated to promoting knowledge of techniques, procedures and strategies in order to ensure use of the highest quality practices in reproductive medicine. The need to develop a set of guidelines was a logical consequence that found its first expression in 1990, when Focus on Reproduction (vol. 1, pp. 10-38) published the first guidelines which were distributed among the membership. Five years later a new, more complete edition with several novel techniques and developments appeared in Human Reproduction (vol. 10, pp. 1246-1271). Both have proved to be invaluable references. Five more years have now passed. The necessity to produce current guidelines for good IVF laboratory practice has provided the strongest motivation. This originated from the increasing awareness that embryologists have a duty to prevent unintentional incidents that might result from poor practice in the laboratory. Therefore, the Embryology Special Interest Group (SIG) undertook to draw up guidelines aimed at giving support and guidance to the laboratory staff. All the aspects required to provide a safe working system were taken into consideration by members of the SIG and their effort produced this document. We hope that it will assist staff in achieving the best clinical outcome for their patients.

Objective. The objective of this study was the determination of sex chromosome aneuploidy frequency in spermatozoa from patients included in an in vitro fertilization (IVF) or intra cytoplasmic sperm injection (ICSI) protocol. Methods. Spermatozoa from nineteen patients, including patients with normal seminal parameters according to World Health Organization (WHO) criteria and patients exhibiting abnormal seminal parameters, were analyzed by dual color fluorescence in situ hybridization (FISH) for aneuploidies of the X and Y chromosomes. Our technique, using only probes for sex chromosomes and not for autosomes, does not discriminate between hyperhaploid and diploid sperm nuclei. The results were analyzed in two different ways: in relation to the semen status, denoted normal or abnormal and with regard to the ability of the sperm to fertilize oocytes when IVF or ICSI was performed. Results. Abnormal semen showed a significant increase in the overall rate of sperm nuclei with XY, XX and YY sex chromosome complements, 1.59% compared to normal semen, 0.78% (p

The rationale for assisted hatching Among the numerous reasons that could explain the low implantation rate of embryos used for in-vitro fertilization (IVF), an impairment of in-vivo hatching has been proposed as a possible cause of implantation failure by Cohen et aI. (1990a). Indeed, this group had shown that implantation rates of human embryos were correlated to the variability of zona pellucida thickness, ranging from 10% for embryos with uniform zonae to 29% for embryos with a thin or irregular zona pellucida (Cohen et aI., 1989). They also reported that micromanipulated embryos taken from couples with male problems implanted at higher rates than intact embryos, and that 25% of human embryos displayed zona pellucida thinning when proceeding from the zygote to the 4-cell stage (Wright et aI., 1990). In vitro, any effect of uterine fluid in digesting the zona pellucida is lacking, and only 25% of human blastocysts achieve hatching in culture (Lindenbergh et aI., 1989; Dokras et at., 1991), even when they look normal and secrete adequate amounts of human chorionic gonadotrophin (HCG) (Woodward, 1993). In vivo, however, how can hatching be impaired? Several hypotheses have been proposed: these include extrinsic factors such as zona pellucida hardening in culture, as described by De Felici and Siracusa (1982) in mice, or intrinsic factors of embryonic or uterine origin. The lytic action of uterine fluid may be impaired in some patients or in some cycles. Likewise, some embryos may be unable to escape from their zonae. Moreover, a thick or hard zona pellucida may hinder the hatching process. Cohen et at. (1990a) reported that 15% of IVF embryos displayed a thick zona pellucida, on average> 15 /-lm. It is more difficult to assess zona pellucida hardness, as did Longo (1981) in mice, by calculating the time of exposure to trypsin necessary to digest the zona pellucida and so detected an increased hardness in ageing eggs. In humans, too, it is apparent that spontaneous zona hardening can occur within 24 h of in-vitro culture, and involves similar changes to the zona as after fertilization. However, this zona hardening does not increase further whereas there is no correlation between zona hardness and zona thickness or patient age (Schiewe et at., 1995a).

Abstract 242 Introduction Hydroxyurea (HU) has proved its efficacy in reducing vaso-occlusive events in patients with SCD and increasing life expectancy. However, effects on fertility in adult males represent a major issue for acceptance and adherence, as HU has been reported to impair spermatogenesis through direct cytotoxic effects and hypogonadism. There are only limited data in the literature in this field and mainly retrospective. Patients and methods 49 adult patients aged 20–52 years with homozygous SCD eligible for first line HU treatment were enrolled after informed consent between June 2010 and April 2012 in this prospective study: HYDREP.The main objective was to compare at Day 0 and 180 of HU treatment the semen parameters according to the WHO (1999) criteria(volume, sperm concentration, viability, forward motility and morphology ). Vaso-occlusive events and other complications, number of transfusions during the 6 months preceding and following HU initiation were recorded. Hematological, biochemical, hormonal parameters, HU dosage were recorded at Day 0, Day 90 and 180. HU was begun at 15mg/Kg dose (Platt *) and modulated following haematological tolerance and renal function. Results and discussion Preliminary results concern the 24/49 patients, who complete the 6 months protocol. A statistically significant impairment in all sperm parameters was observed, after a 6 months HU therapy, with great variations between individuals, which are to be explained by complementary tests. These preliminary deleterious findings must be interpreted with caution as in our experience 1) spermatogenesis may recover after drug withdrawal 2) Repeated vaso-occlusive events are also potentially deleterious on fertility (Berthaut **)and 3)this study must be completed with a survey on effective fertility seen in men treated with HU. Thus, regarding to the great benefit offered by HU on prognosis allowing a real “life project” in young symptomatic adults with SCD, it is worth providing each patient of the most accurate information on fertility as an essential prerequisite for their adhesion to HU treatment. Conclusion Sperm cryopreservation is advised before beginning a treatment with HU. Prospective follow up studies of HU male cohort regarding fertility are warranted. Health education has a major role to play to increase HU compliance especially in view of these results. Disclosures: No relevant conflicts of interest to declare.