Preimplantation genetic diagnosis in an HIV-serodiscordant couple carrier for sickle cell disease: lessons from a case report.

Since 1999, the Erasme Hospital Fertility Clinic has carried a special programme for patients with HIV seropositivity. The philosophy of the programme is to give access to these patients in a secure environment to the same technological facilities available to any other patients. Many of these patients being native from sub-Saharan countries, they are often sickle cell disease (SCD) carriers, a common autosomal recessive disorder in these regions, and a severe affection in homozygotes. We hereby report, for the first time, the birth of a healthy sickle haemoglobin (HbS) heterozygous baby after preimplantation genetic diagnosis (PGD) for SCD in an HIV-serodiscordant couple of HbS mutation carriers with longstanding infertility. The prospective mother was 35 years old and HIV positive with an undetectable viral load under highly active antiretroviral therapy. One carrier embryo was transferred and resulted in the birth of a healthy HbS carrier baby girl. Despite stimulation difficulties, sometimes described in HIV patients, PGD represents an interesting additional technology, especially in populations where the coexistence of both diseases is frequent. PGD could even be preferred to prenatal diagnosis for couples of HbS carriers if the woman is HIV positive, as invasive prenatal samplings carry a risk of materno-foetal viral transmission.

Immune hyperactivation of HIV-1-infected T cells mediated by Tat and the CD28 pathway.

Human immunodeficiency virus-type 1 (HIV-1) infection is characterized by a chronic state of immune hyperactivation in patients. Infection of human peripheral blood lymphocytes with HIV-1 in vitro resulted in increased interleukin-2 (IL-2) secretion in response to T cell activation via the CD3 and CD28 receptors. Expression of the HIV-1 transactivator Tat recapitulated this phenotype and was associated with increased IL-2 secretion in response to costimulation with CD3 plus CD28. IL-2 superinduction by Tat occurred at the transcriptional level, was mediated by the CD28-responsive element in the IL-2 promoter, and was exclusively dependent on the 29 amino acids encoded by the second exon of Tat.

[Preimplantation genetic diagnosis (PGD): the Erasme Hospital experience]. / Le diagnostic génétique préimplantatoire (DPI): l'expérience de i'Hôpital Erasme.

The clinical activity of the preimplantation genetic diagnosis (PGD) at Erasme Hospital was carried out since September 1999 for a 47,XYY patient. Up to 31 December 2007, 79 PGD cycles were carried out (45 couples) for either chromosomal structural abnormalities (robertsonian and reciprocal translocations, pericentric inversion, deletion) (n = 41), chromosomal numerical abnormalities (47,XXY, 47,XYY, 45,X/46,XX) (n = 10), aneuploidy screening for recurrent miscarriages or multiple in vitro fertilization failures (n = 10), autosomal recessive diseases (cystic fibrosis and sickle cell anaemia) (n = 12) or X-linked disorders (n = 6). A total of 475 embryos were biopsied for genetic analysis. Unaffected embryos were transferred in 58 cycles, resulting in 22 pregnancies, including fifteen clinical pregnancies. Up to now, 9 babies were born and 3 pregnancies are still ongoing. After a learning curve, our current PGD efficiency shows a total pregnancy rate per transfer of 60.0% and an implantation rate of 28.6%. Each PGD result was confirmed by prenatal or postnatal diagnosis. Our data demonstrate that PGD is a valid technique to allow couples at high risk of transmitting a genetic abnormality to increase their chances of a healthy pregnancy, but considering its complexity, patients must be counselled and selected rigorously.

Acetylation of the HIV-1 Tat protein by p300 is important for its transcriptional activity.

The human immunodeficiency virus 1 (HIV-1) Tat protein activates transcriptional elongation by recruiting the positive transcription elongation factor (pTEFb) complex to the TAR RNA element, which is located at the 5' extremity of all viral transcripts [1-3]. Tat also associates in vitro and in vivo with the transcriptional coactivator p300/CBP [4-6]. This association has been proposed to recruit the histone acetyltransferase (HAT) activity of p300 to the integrated HIV-1 promoter. We have observed that the purified p300 HAT domain acetylates recombinant Tat proteins in vitro and that Tat is acetylated in vivo. The major targets of acetylation by p300 are lysine residues (Lys50 and Lys51) in the arginine-rich motif (ARM) used by Tat to bind RNA and for nuclear import. Mutation of these residues in full-length recombinant Tat blocked its acetylation in vitro. Furthermore, mutation of these lysine residues to arginine markedly decreased the synergistic activation of he HIV promoter by Tat and p300 or by Tat and cyclin T1. These results demonstrate that acetylation of Tat by p300/CBP is important for its transcriptional activation of the HIV promoter.

Interaction between cyclin T1 and SCF(SKP2) targets CDK9 for ubiquitination and degradation by the proteasome.

CDK9 paired with cyclin T1 forms the human P-TEFb complex and stimulates productive transcription through phosphorylation of the RNA polymerase II C-terminal domain. Here we report that CDK9 is ubiquitinated and degraded by the proteasome whereas cyclin T1 is stable. SCF(SKP2) was recruited to CDK9/cyclin T1 via cyclin T1 in an interaction requiring its PEST domain. CDK9 ubiquitination was modulated by cyclin T1 and p45(SKP2). CDK9 accumulated in p45(SKP2-/-) cells, and its expression during the cell cycle was periodic. The transcriptional activity of CDK9/cyclin T1 on the class II major histocompatibility complex promoter could be regulated by CDK9 degradation in vivo. We propose a novel mechanism whereby recruitment of SCF(SKP2) is mediated by cyclin T1 while ubiquitination occurs exclusively on CDK9.

[Single embryo transfer : impact of new Belgian legislation on the results of the Clinic of Fertility of the Erasme Hospital]. / Le transfert d'un embryon unique : impact de la nouvelle loi beige sur les résultats du Centre de Fécondation in vitro (FIV) de l'Hôpital Erasme.

With the progress made in the treatments of assisted reproduction, implantation and pregnancy rates have increased. This evolution has led to increase the rates of multiple pregnancies in the general population. Considering maternal and fetal risks related to multiple pregnancies it was necessary to reduce their incidence. Several efforts have been tried, in particular the limitation of the number of embryos transferred to 2. This reduced the incidence of triplets but that of twin remained unchanged, which convinced the clinicians of the need to reduce further the number of embryo transfer. In Belgium a new policy of transfer was established by a law introduced since the 01/07/2003 aiming to reduce the costs related to the twin pregnancies and to increase the reimbursement of IVF treatments. We have studied the impact of this policy on the results at the clinic of Erasme. Two periods were compared : from 01/01/2001 to 30/06/2003 where the majority of the transfers was transfers of 2 embryos (56.8 %) and from 01/07/2003 to 31/12/2004 where the majority of the transfers was transfers of a single embryo (53.7 %) (p < 0.001). The rates of single embryo transfer were 12.5 % and 53.7 % respectively (p < 0.001). The rates of clinical pregnancies were 33.2 % and 27.3 % respectively (p < 0.001), on the other hand the percentage of twin pregnancies has strongly decreased from 29.9 % to 11.4 % (p < 0.001). The rate of frozen embryos has increased from 22 % policy seems to achieve its goals to the detriment of a reduction of the success rates. Nevertheless, the increase in the number of frozen embryos should allow, after thawing and transfer, to compensate at least partially this reduction of the pregnancy rate.

The periodic down regulation of Cyclin E gene expression from exit of mitosis to end of G(1) is controlled by a deacetylase- and E2F-associated bipartite repressor element.

The expression of cyclin E and that of a few other bona fide cell cycle regulatory genes periodically oscillates every cycle in proliferating cells. Although numerous experiments have documented the role of E2F sites and E2F activities in the control of these genes as cells exit from G(0) to move through the initial G(1)/S phase transition, almost nothing is known on the role of E2Fs during the subsequent cell cycles. Here we show that a variant E2F-site that is part of the Cyclin E Repressor Module (CERM) (Le Cam et al., 1999b) accounts for the periodic down regulation of the cyclin E promoter observed between the exit from mitosis until the mid/late G(1) phase in exponentially cycling cells. This cell cycle-dependent repression correlates with the periodic binding of an atypical G(1)-specific high molecular weight p107-E2F complex (Cyclin E Repressor Complex: CERC2) that differs in both size and DNA binding behaviors from known p107-E2F complexes. Notably, affinity purified CERC2 displays a TSA-sensitive histone deacetylase activity and, consistent with this, derepression of the cyclin E promoter by trichostatin A depends on the CERM element. Altogether, this shows that the cell cycle-dependent control of cyclin E promoter in cycling cells is embroiled in acetylation pathways via the CERM-like E2F element.

Subtyping of human immunodeficiency virus isolates with a panel of monoclonal antibodies: identification of conserved and divergent epitopes on p17 and p25 core proteins.

We have investigated the feasibility and significance of subtyping of human immunodeficiency virus (HIV) isolates with monoclonal antibodies (mAb) raised against the core proteins of HIV. A panel of 37 mAb tested for reactivity with HIV1 oligopeptides was used to analyse the antigenic relatedness among 14 HIV isolates which included 12 isolates of HIV1 from different geographical origins and 2 isolates of HIV2. Three out of these 37 mAb reacted with conserved epitopes expressed by all 14 HIV isolates tested. These reagents which included 2 mAb reacting with the 285-310 amino acid sequence of p25 and 1 mAb reacting with an epitope of p25 not mapped by the peptides' approach, also reacted with a non-human primate lentivirus. Five mAb reacting either with the 11-25 or 121-132 amino acid sequences of p17 or the 302-320 amino acid sequence of p25 reacted with strain-specific epitopes. The other 29 mAb reacted with polymorphic epitopes and thereby define subfamily and subtype-specific markers.

Clonal analysis of murine B cell response to the human immunodeficiency virus type 1 (HIV1)-gag p17 and p25 antigens.

The antigenicity of HIV-gag p17 and p25 proteins was analyzed using a panel of 52 monoclonal antibodies (mAb) derived from 17 independent fusion experiment protocols performed in 12 different laboratories. These mAb were tested for their capacity to bind peptides corresponding to sequences of HIV1-BRU-gag p17 and p25. Thirty-five overlapping peptides (P1 to P35) totally covering the p17 and p25 proteins were used. This study allowed us to identify four immunodominant regions inducing B cell response, two on p17 corresponding to P2 and P13 (amino acids 11-25 and 121-132, respectively) and two on p25 corresponding to P21 and P28-P29-P30 (a.a. 201-218 and 285-320 respectively). According to secondary structure predictions, peptides P2 and P21 contained hydrophilic alpha helix folded regions whereas P13 sequence presented a beta turn propensity. These regions and the P28-30 region were also predicted to be easily accessible to mAb. Several other p25-derived peptides: P15 (a.a. 142-156), P16 (a.a. 148-162), P19 (a.a. 176-192), P22 (a.a. 219-233) and P23 (a.a. 233-253) were recognized by mAb. No p17-derived peptide other than P2, P13 and P12 (a.a. 111-123) was found to react with mAb. Cross-blocking studies between mAb, suggested the existence of more than four distinct epitopic areas on p17 and eight on p25.

Structural and Functional Properties of HIV-1(GER) TAR Sequences.

Sequencing of HIV-1(GER) long terminal repeat (LTR) has demonstrated, for the first time in an HIV-1 primary isolate, a TAR duplication referred to as TAR1 (nucleotides +1 through +68) and TAR2 (nucleotides +69 through +136). This TAR duplication is stable during replication of HIV-1(GER) isolate in CEM cells. Analysis of LTR-CAT reporter constructs demonstrated that under Tat transactivation the HIV-1(GER)/LTR (containing TAR1 and TAR2) was expressed at a higher level than a similar construct (HIV-1(GER)DeltaTAR) containing a single TAR sequence. Among the two transcription initiation sites found in the HIV-1(GER)/LTR, only the most 5' start site was shown to be functionally active. The predicted secondary structure of the 5'-end mRNAs of HIV-1(GER) suggests it may fold into a double TAR hairpin which resembles that of HIV-2. Finally, HIV-1(GER) Tat protein shows primary sequence similarity with Tat proteins from other isolates of HIV-1 and is apparently unrelated to HIV-2 Tat proteins. This work provides the first evidence of a TAR sequence duplication in HIV-1 which increases the efficiency of transactivation by Tat. Copyright 1996 S. Karger AG, Basel

The expression of a small fraction of cellular genes is changed in response to histone hyperacetylation.

Posttranslational modifications of histones in chromatin are emerging as an important mechanism in the regulation of gene expression. Changes in histone acetylation levels occur during many nuclear processes such as replication, transcriptional silencing, and activation. Histone acetylation levels represent the result of a dynamic equilibrium between competing histone deacetylase(s) and histone acetylase(s). We have used two new specific inhibitors of histone deacetylase, trichostatin A (TSA) and trapoxin (TPX), to probe the effect of histone hyperacetylation on gene expression. We confirm that both drugs block histone deacetylase activity and have no detectable effects on histone acetylation rates in human lymphoid cell lines. Treatment with either TSA or TPX results in the transcriptional activation of HIV-1 gene expression in latently infected cell lines. In contrast, TSA and TPX cause a rapid decrease in c-myc gene expression and no change in the expression of the gene for glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Using differential display to compare the differences in gene expression between untreated cells and cells treated with TSA, we found that the expression of approximately 2% of cellular genes (8 genes out of approximately 340 examined) changes in response to TSA treatment. These results demonstrate that the transcriptional regulation of a restricted set of cellular genes is uniquely sensitive to the degree of histone acetylation in chromatin.

Plasma homocysteine after insulin infusion in type II diabetic patients with and without methionine intolerance.

BACKGROUND: A high prevalence of hyperhomocysteinemia has been reported in type II diabetic patients with documented vascular disease; hence the hypothesis that hyperhomocysteinemia may contribute to overall mortality in diabetic patients. The link between insulin and homocysteine metabolism has not been completely clarified yet; in particular, only few data are available on the effects of insulin in vivo on homocysteine metabolism in the presence of abnormalities of sulphur amino acid metabolism (methionine intolerance). MATERIALS AND METHODS: To establish whether methionine intolerance and which of its determinants could influence total plasma homocysteine in response to insulin infusion in vivo in type II diabetic patients, we submitted 18 patients (Group A) with normal and 18 patients with abnormal (hyperhomocysteinemia) (Group B) response to oral methionine load to a glucose/clamp study. At time 0, and 30, 60 and 120 minutes after hyperinsulinemia, homocysteine and methionine plasma levels were assessed. In order to evaluate the cause of methionine intolerance, all patients were assayed for fasting homocysteine-cysteine ratio (as a marker of suspected heterozygosis for cystathionine-beta-synthase deficit), MTHFR C (677)T status and homocysteine-related vitamin status (serum vitamin B (6) [PLP], vitamin B (12) and folate). RESULTS: After hyperinsulinemia, plasma methionine was reduced (by about - 30 % at 120 minutes vs. basal values) within both groups, whereas tHcy tend to decrease in group A following insulin administration (up to - 6.6 +/- 3.6 % vs. basal values at 120 minutes) with a significantly higher variability, while in patients with "methionine intolerance" (group B) tHcy tended to increase (up to + 29.05 +/- 8.3 % vs. basal values at 120 min from the clamp). Serum folic acid (7.45 +/- 2.8 vs. 4.82 +/- 2.5 nmol/L, p < 0.05), Vit. B (12) (348 +/- 78 vs. 242 +/- 65 pmol/L, p < 0.05) and PLP (84.1 +/- 23.6 vs. 50.6 +/- 32.4 nmol/L; p < 0.01) were significantly higher in group A than in group B; PLP levels significantly correlated with homocysteine after 4 h methionine load (n = 36; r = - 0.327, p < 0.05); group A showed also a significantly lower prevalence of suspected heterozygosis for cystathionine-beta-synthase deficit (1/18 [11.1 %] vs. 5/18 [33.3 %], p < 0.05) and MTHFR T allele presence (4/18 [22.2 %] vs. 11/18 [61.1 %], p < 0.01). A stepwise regression analysis with tHcy plasma level variations (event A = reduction; event B = increase) as the dependent variable showed that low serum folate and PLP levels and presence of MTHFR T allele were the variables associated with insulin-induced tHcy increase. CONCLUSIONS: Methionine intolerance may influence the effect of insulin administration on plasma homocysteine in patients affected by type 2 diabetes. To prevent a possible acute (and repeated) hyperhomocysteinemia due to insulin administration in cases of methionine intolerance, it may be useful to assess the presence of methionine intolerance (tHcy after oral methionine loading) and Hcy-related vitamin status in all patients due to be subjected to insulin therapy.

Nitric oxide in preeclampsia: lack of evidence for decreased production.

The purpose of our study was to determine the involvement of the L-arginine-NO system in preeclampsia. We studied 26 patients with preeclampsia and 27 normotensive pregnancies. Maternal and cord plasma, urine and amniotic fluid were assayed for nitric oxide metabolites (nitrite and nitrate) using the Griess reaction. Sections of placenta and fetal membranes were immunostained with polyclonal anti-endothelial and anti-neuronal nitric oxide synthase antibodies. The concentration of nitrate in the amniotic fluid of preeclamptic patients (median 10.3 mumol/mg creatinine) was significantly higher (P < 0.001) than in the normotensive group (5.6 mumol/mg creatinine). Nitrate concentrations in maternal and cord plasma and in urine were similar in the two groups. Endothelial cells of the villi of preeclamptic placentas showed a higher positivity in endothelial nitric oxide synthase immunostaining with respect to normotensive controls. Our results indicate that feto-placental NO production is not reduced in preeclampsia. In contrast, the increased concentrations of NO metabolites in amniotic fluid and the positive immunostaining of endothelial nitric oxide synthase in the placental villi suggest that the placental L-arginine-NO system is up-regulated in preeclampsia.

[Modification of in vitro insemination techniques in the treatment of severe male factors in assisted reproduction]. / Modificazione delle procedure di inseminazione in vitro nel trattamento dei gravi fattori maschili nella riproduzione assistita.

The aim of this study is to detect qualified in vitro insemination techniques in the treatment of the severe oligoasthenotheratospermia which is defined as total motile count in the pretreatment samples (< 5 x 10(6) with > 50% of abnormal morphology). These patients have taken part in the in vitro insemination program of the Assisted Reproduction Unit of the 2nd Department of Obstetrics and Gynecology at Rome University "La Sapienza" during a period between June and December 1995. Several modifications of the standard in vitro techniques have been developed such as: mechanical decumulation of the oocytes, reduction of the volume of culture medium, increase of spermatozoa and oocyte concentration at the moment of insemination. A good fertilization rate was achieved (33%) as regard to the semen sample and procedures utilized. Twelve Ets were performed and 4 clinical pregnancies (25% per patients and 33% per transfer) were achieved. These data demonstrate that by the modification of standard laboratory methods for in vitro insemination, a good fertilization rate and a high clinical pregnancy rate can be achieved in cases of severe male factor infertility without having to resort to micromanipulation techniques.

[Impact of the introduction of intracytoplasmic sperm injection (ICSI) on the treatment of severe male sterility]. / L'impact de l'introduction de la microinjection (ICSI) sur le traitement de la sterilite a composante masculine severe.

The introduction of the Intracytoplasmic Single Sperm Injection (ICSI) has been a turning point for the treatment of severe male infertility. ICSI allowed not only to reduce fertilization failure from 35% to 0.7% but created at the same time the opportunity for a group of patients with extremely low sperm counts to procreate. The discovery that breaking the tail of the spermatozoon prior to the injection was the most important step is at the origin of major improvements: fertilization increased from 22% to 77%, pregnancy rate from 16% to 54% and the implantation rate from 7.4% to 26%. From October 1994 to April 1999, 835 ICSI cycles were performed and resulted in 312 ongoing pregnancies (37%), fertilization rate was 75%, with a fertilization failure of only 0.7%. The use of ICSI and IVF on sibling oocytes for semen samples with doubtful fertilizing ability clearly illustrated the superiority of ICSI. No fertilization failures occurred after ICSI and the fertilization rate was 76% versus 27.8% (P < 0.01). Similar benefit of ICSI was shown for crytozoospermia up to then a hopeless situation. A total of 26 pregnancies were obtained out of 87 cycles with a fertilization rate of 58.8%. Similar results were obtained when ICSI was combined with testicular sperm, 20 pregnancies occurred after from 46 transfers (43%) including cycles with cryopreserved testicular sperm. It is now clear that ICSI is the method of choice for the treatment of severe male infertility.

[Diminishing the risk of multiple pregnancies in in vitro fertilization: from selective transfer of two embryos to that of one blastocyst?]. / Diminuer le risque de grossesse multiple en fecondation in vitro: du transfert selectif de 2 embryons a celui d'un seul blastocyste?

The risk of multiple pregnancy after IVF needs to be drastically reduced. Several policies can be applied including the transfer of a maximum of three embryos to all patients, the fertilization of a maximum of three oocytes or a selective reduction of the number of transferred embryos. The first policy previously applied at the Fertility Clinic at Erasme Hospital until 1996, transferred two good quality embryos to patients with at least three good embryos. If this policy demonstrated that patients with two transferred embryos had similar chances of pregnancies compared to patients with three transferred embryos, it failed to sufficiently decrease the number of multiple pregnancies. The second policy applied since 1997, transferring a maximum of two average or good embryos to all patients aged under 35 years and with less than 3 previous attempts, demonstrated that while preserving the chances of pregnancy for these patients, it decreased by 20% the number of multiple pregnancies and almost eliminated triplets. With the improvement of culture media, it is now possible to culture embryos in vitro for a longer period and therefore transfer embryos with proven viability at a time corresponding more to in vivo physiological conditions. The implantation rates for these embryos, for patients with at least 4 previous attempts can reach 40%. If these results persist, it would be possible to transfer blastocysts to all patients and perhaps move on to the replacement of a single embryo, a policy that will practically eradicate all multiple pregnancies.

[The gynecology-obstetrics department]. / Le Service de Gynécologie-Obstétrique.

The scientific and clinical activities of the Department of Obstetrics and Gynaecology have involved the three main subdivisions: the gynecological surgery, the obstetrics and fetal medicine, the endocrinology and the reproductive medicine. Minimal invasive surgery including laser assisted laparoscopy or robotic assisted surgery has been particularly developed. Endometriosis, a frequent and sometimes particularly invasive disease, and oncologic surgeries have been developed in collaboration with the digestive surgery department. The department has also contributed to the comprehension and treatment of prenatal pathologies such as premature labor and deliveries or the gestational diabetes. The department has supported the development of techniques to study the fetal well-being in utero: the prenatal echography, the chorionic villous sampling, the amniotic puncture or the cordocentesis for prenatal genetic diagnosis or fetal infectious contaminations, the CMV transmission more specifically. In endocrinology and reproductive medicine, the department has mainly developed the in vitro fertilization techniques. The prolonged embryo culture, the study of preimplantation embryo metabolism, the preimplantation genetic diagnosis and the cryopreservation of ovarian fragments to preserve fertility in women undergoing oncologic treatments represent the more recent developed topics. Finally, the security of viral transmission in assisted procreation and the treatment of these patients with chronic viral diseases (Hepatitis C or HIV) are another domain with important scientific activity.

[Aspects of scientific research and technical progress in human fertility]. / Aspects de recherche scientifique et progres techniques en fertilite humaine.

The development of an outstanding in vitro fertilization program greatly benefits from the contribution of research because it remains an unfailing source of questions on human reproduction, as much in the fields of physiology and pathology as in those of psychology and sociology. This paper shows five major themes that are tackled by the laboratory of biology and psychology of human fertility and the Fertility Clinic, whether it's endocrinology (the ovarian renin and angiotensin regulation), cellular metabolism (embryo metabolism), genetics (preimplantation genetic diagnosis) or cancerology (ovarian tissue conservation before or after chemo- or radiotherapy), all of these are crossed by the fifth (the psychological and ethical aspects of in vitro fertilization) which gives a human dimension to the biological work, since it's a very special biology that it's our own reproduction, the very base of the specie's survival.

[In vitro fertilization at the Erasme Hospital: 10 years and 1000 pregnancies later...]. / La fecondation in vitro a l'Hopital Erasme: 10 ans et 1000 grossesses plus tard....

This contribution summarize ten years of in vitro fertilization of clinical work. Activity growth, improvements of results (mean fertilization rate increased from 45% to 58%, fertilization failure dropped from 18% to 7%, pregnancy chances gains 9% to reach 44% per trial) and new treatments possibilities (severe male infertility) thanks to the ICSI technic were the major characteristics of this last ten years. The original anonymous oocyte donation program with donors permutation initiated as soon as 1990 has imposed itself due to it's exceptional efficiency with a pregnancy rate of 95% per oocyte pick up on a population of 46 donors and 145 recipient cycles. Thanks to the large population studied (4028 cycles, 1071 pregnancies), the tendencies in human fecundity (impact of age) and the risks linked to multiples pregnancies could be highlighted, stressing the importance of future developments presented in the other contributions following this general presentation of results.