Orthotopic and heterotopic ovarian tissue transplantation.

BACKGROUND: Transplantation of ovarian tissue is, at present, the only clinical option available to restore fertility using cryopreserved ovarian tissue. More than 30 transplantations of cryopreserved tissue have been reported, and six babies have been born, worldwide, following this procedure. Despite these encouraging results, it is essential to optimize the procedure by improving the follicular survival, confirming safety and developing alternatives. Here, we review the different factors affecting follicular survival and growth after grafting. METHODS: Relevant studies were identified by searching Pubmed up to January 2009 with English language limitation. The following key words were used: (ovarian tissue or whole ovary) AND (transplantation) AND (cryopreservation or pregnancy). Using the literature and personal experience, we examined relevant data on the different exogenous and clinical factors affecting follicular development after grafting. RESULTS: Clinical factors such as the patient's age and the transplantation sites influenced the lifespan of the graft. A heterotopic transplantation site is not optimal but offers some advantages and it may also promote the hormonal environment after a combined heterotopic and orthotopic transplantation. Exogenous factors such as antioxidants, growth factors or hormones were tested to improve follicular survival; however, their efficiency regarding further follicular development and fertility potential remains to be established. CONCLUSION: Additional evidence is required to define optimal conditions for ovarian tissue transplantation. Alternatives such as whole ovary or isolated follicles transplantations require further investigation but are likely to be successful in humans in the future.

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.

[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.

[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.

Options to preserve fertility before oncological treatment: cryopreservation of ovarian tissue and its clinical application.

Chemotherapy and radiotherapy induce premature ovarian failure in many patients treated with these methods for oncological and benign diseases. This paper reviews the risk of developing premature ovarian failure according to the types of treatment as well as the different options to preserve fertility. We focus mainly on the cryopreservation of ovarian tissue procedure and we report here the second world-wide spontaneous pregnancy after cryopreserved ovarian tissue transplantation.

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.

[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.

Preimplantation genetic diagnosis (PGD), a collaborative activity of clinical genetic departments and IVF centres.

Preimplantation genetic diagnosis (PGD) requires the combined efforts of geneticists and workers in the field of reproductive medicine. This was studied on the basis of a questionnaire, sent to 35 members of the PGD Consortium of the European Society of Human Reproduction and Embryology (ESHRE). A reply was obtained from 20 centres. They represent the majority of activities in the field of PGD in the world. It is obvious that many of the activities (in vitro fertilisation, embryo culture and biopsy) take place in IVF units while others (counselling and diagnosis) are the responsibility of genetic diagnostic centres. The distances between both units vary considerably. In all but one centre sex determination is offered. Aneuploidy screening is offered in 13 out of 20 centres. PGD of translocations and other structural chromosome abnormalities is offered in all but one centre. The number of monogenic diseases offered varies considerably. In comparison to prenatal diagnosis PGD is more expensive. The majority of these costs are due to the IVF or ICSI procedure. The charges for PGD vary between about 600 euro and 4000 euro. In 16 out of 20 centres the parents to be must sign an informed consent form.

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.

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.

Evidence of reduced single-stranded testicular sperm DNA from obstructive azoospermic men after 3 days of in-vitro culture.

The aim of the present study was to verify whether culturing testicular tissue, to obtain a higher percentage of motile spermatozoa and a better post-thaw recovery rate, affected the ratio between single/double-stranded sperm DNA and, consequently, DNA sensitivity to damage. Testicular biopsy samples from men with obstructive and secretory azoospermia, candidates for assisted reproductive treatment, were cultured for 72 h. The percentage of motile spermatozoa and the single/double stranded DNA ratio were assessed on the day of retrieval (day 0) and again on day 3. The single/double stranded DNA ratio was measured by the acridine orange (AO) staining method. Spermatozoa were classified as green (double-stranded chromatin) or red fluorescing (single-stranded chromatin). In obstructive azoospermia, median motility was 22% (range 10-44%) on day 0 and 50% (range 38-63%) on day 3 (P < 0.01). The median percentage of red stained spermatozoa was 53.5% (range 0.1-88%) on day 0 and 20% (range 2.7-99.9%) on day 3 (P < 0.05). No changes were observed in secretory azoospermia. The culture procedure from obstructive azoospermia not only increased the post-thaw recovery rate, as previously observed, but also reduced the portion of spermatozoa containing single-stranded DNA, thereby increasing the availability of double-stranded DNA spermatozoa for ICSI use.

Amino acids promote human blastocyst development in vitro.

Supplementation of culture media with amino acids has been shown to benefit preimplantation embryo development in several species. This randomized study analysed the in-vitro development of human embryos obtained after IVF in the presence or absence of a combination of amino acids from the 2- to 4-cell stage to the blastocyst stage. A total of 129 human embryos was randomly distributed between three serum-free chemically defined sequential media: (i) glucose-free Earle's balanced salt solution (EBSS) with glutamine (Gln) prior to morula stage, supplemented with glucose for blastocyst formation; (ii) glucose-free EBSS with glutamine and non-essential amino acids (AA) for cleavage stage development, and supplemented with all 20 AA for blastocyst formation (Earle's+AA); and (iii) a sequential commercial medium containing amino acids (K-SCIM). Embryos were individually cultured for successive periods of 24 h. On day 6 of development, blastocysts were differentially labelled and the numbers of trophectoderm and inner cell mass cells, mitoses and dead cells were examined. Blastocyst development was similar for the three sequential media. The mixture of AA significantly increased total blastocyst cell numbers from 61.8 +/- 4.2 with Earle's+Gln to 99.3 +/- 8.4 with Earle's+AA and 100.2 +/- 9.4 with K-SCIM (P = 0.005). This increase was present in both the trophectoderm and inner cell mass lineages (P < 0.02). Furthermore, the dead cell index was significantly lower with Earle's+AA (P = 0.047).

Increased sperm motility after in-vitro culture of testicular biopsies from obstructive azoospermic patients results in better post-thaw recovery rate.

The objective of this study was to optimize the use of testicular biopsies in 14 patients with obstructive azoospermia. Testicular specimens were retrieved from six patients (group I) and cultured at 32 and 37 degrees C for up to 20 days; changes in percentage motile spermatozoa were compared. In four men of group I, one portion of the specimen was frozen at retrieval, and changes in post-thaw motility after 24 h of culture at 37 degrees C were recorded. In the other eight patients (group II), testicular specimens were frozen at retrieval and after 72 h culture at 37 degrees C. Pre and post-freezing motility and post-thaw recovery rate were compared. No significant differences were observed until day 8 in the improvement of motility between 32 and 37 degrees C in-vitro culture. Maximum motility was reached, under both conditions, between 48 h and 72 h. Post-thaw 24 h culture at 37 degrees C of specimens frozen at retrieval did not improve motility; however, 72 h pre-freezing culture significantly improved initial motility (P: < 0.01), post-thaw motility (P: < 0.01) and post-thaw recovery rate (P: < 0. 001). The higher recovery rate of samples frozen 3 days after retrieval allows more economical use of the tissue that is available.

Comparison of ethylene glycol, 1,2-propanediol and glycerol for cryopreservation of slow-cooled mouse zygotes, 4-cell embryos and blastocysts.

The aim of the study was to analyse the toxicity, the osmolar and cryoprotective activity of ethylene glycol (ETG) in terms of survival rate (SR), cleavage rate (CR) and expanded blastocysts percentage (EBP) of mouse embryos. Early mouse embryos and blastocysts were slowly cooled with ETG, 1,2-propanediol (PROH) or glycerol, and thawed. The Van t'Hoff curve for 1.5 mol/l ETG showed recovery of initial volume within 4 min. No differences were observed in CR and EBP of ETG-exposed compared with non-exposed mouse zygotes. The SR of zygotes frozen with PROH was significantly better than with ETG (92% and 60% respectively; P < 0.01), and a significantly better EBP was achieved for blastocysts frozen with glycerol compared with ETG (75% and 50% respectively; P < 0.05). For 4-cell stage embryos, no differences were observed in SR and EBP between ETG and PROH. Higher EBP was observed for 4-cell stage embryos (53%) frozen with ETG compared with pronucleate stage (19%) and blastocysts (48%). Low toxicity, good SR and EBP were observed for mouse embryos frozen with ETG, the best results being obtained at the 4-cell stage. At other embryonic stages, PROH and glycerol respectively seemed to provide better results.

Re-analysis by fluorescence in situ hybridisation of spare embryos cultured until Day 5 after preimplantation genetic diagnosis for a 47, XYY infertile patient demonstrates a high incidence of diploid mosaic embryos: a case report.

Mosaicism in 4-8-cell human embryos analysed by fluorescence in situ hybridisation (FISH) has been widely reported, but few studies have addressed the incidence of mosaicism in more advanced embryonic stages. In the present study we analysed spare human embryos in a case of preimplantation genetic diagnosis (PGD) for increased risk of aneuploidy because of an infertile 47,XYY man. After replacement of two embryos typed as 1818XX at PGD, six spare embryos (not frozen because of their low quality) were re-analysed on Day 5 for PGD confirmation. Out of five embryos typed as 1818XY at PGD, four were diploid mosaic (DM) and one was normal in all cells. The sixth embryo, typed as 18XYY/1818181818X at PGD, was a DM. In spite of the bias of our small series of morphologically low-quality embryos, the surprisingly high proportion of mosaics (which confirms previous findings) questions the validity of PGD, but supports the strategy of transferring only the embryos where two blastomeres gave normal and concordant results at PGD. More data are required to understand the clinical significance of early diploid mosaicism (and its impact on implantation rate) and to determine whether some diploid mosaic embryos might be considered safe for transfer.

[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.

[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.

[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.