Cryopreservation of human failed maturation oocytes shows that vitrification gives superior outcomes to slow cooling.

This study investigated whether failed maturation oocytes could be used to evaluate different cryopreservation procedures. A total of 289 failed maturation oocytes (GV and MI stages), obtained from 169 patients undergoing IVF treatment (mean age 33.84±5.0) were divided into two different slow-cooling groups (1.5 mol/l 1,2-propanediol+0.2 mol/l sucrose in either NaCl (group A) or choline chloride (ChCl) (group B) based cryopreservation solutions) and one vitrification group (15% ethylene glycol+15% dimethyl sulphoxide). Survival rate, in vitro maturation (IVM) rate, fertilization and developmental rate of cryopreserved oocytes were assessed. Regardless of the stage at which cryopreservation was performed (GV+MI), the slow cooling with ChCl based medium always gave significantly lower survival rate than the slow cooling in NaCl based medium (p=0.01) and vitrification (p<0.001). An extended study also showed statistically reduced survival rate between slow-cooling NaCl based medium and vitrification (p<0.05). Global results of in vitro maturation and fertilization showed worse results between both slow-cooling NaCl and ChCl based media versus vitrification. In conclusion, for oocytes that had failed to mature, vitrification gave better survival, maturation, fertilization and also cleavage rates than the slow-cooling protocols. Four cells embryos were obtained only from vitrified in vitro matured MI oocytes.

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.

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.

[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.

The outcome of cryopreserved human embryos after intracytoplasmic sperm injection and traditional IVF.

OBJECTIVE: Our objective was to analyze the outcome of cryopreserved embryos obtained after intracytoplasmic sperm injection (ICSI) and in vitro fertilization (IVF) in terms of survival rate, implantation rate (IR), total and clinical pregnancy rate (PR) in a retrospective, comparative study. METHODS: Three hundred seventy-five IVF and 463 ICSI surnumerary cleaved embryos, frozen on Day 2 with 1,2-propanediol, were thawed. RESULTS: Thirty-two percent of the thawed IVF embryos survived and 11 pregnancies (8 clinical) were obtained from 68 transfers (16.1%). Fourty-seven percent of the ICSI embryos survived, with 19 pregnancies (18 clinical) from 116 transfers (16.4%). The IR was 8.5% (8/94) in IVF cycles and 10.8% (20/185) in ICSI cycles. CONCLUSIONS: A significantly better survival rate of ICSI embryos was observed but with no difference in PR, preclinical, and clinical abortion rate, or IR.

Randomized autocontrolled comparison of the embryo culture performance of Nunc and Falcon petri dishes.

PURPOSE: Our purpose was to compare the embryo culture performance of two types of petri dishes (Nunc and Falcon). METHODS: Mouse zygotes were cultured up to the expanded blastocyst stage in both types of dishes. The oocytes from 50 in vitro fertilization cycles were randomly divided between the two types of dishes. Fertilization, cleavage, and embryo quality were compared. Oocytes from another 50 cycles were all cultured at random in either type of dish. Pregnancy and implantation rates were compared between the two types. RESULTS: Of 91 mouse zygotes, 81 cleaved to two-cell-stage embryos, and 64 became expanded blastocysts in Falcon dishes; of 99 zygotes, 81 cleaved to two-cell-stage embryos and 66 became expanded blastocysts in Nunc dishes. Of 248 oocyte-cumulus complexes (OCC), 145 fertilized in Falcon dishes, and of 269 OCC, 175 fertilized in Nunc dishes. The high quality embryo ratio was 51 out of 118 in Falcon dishes, not different from that in Nunc dishes, 58 out of 139. In Falcon dishes 72 out of 118 embryos were at least at the four-cell stage after 45 hr, versus 70 out of 139 in Nunc dishes. Twenty-three clinical pregnancies were obtained in the first 50 cycles with sibling oocytes. In the second group, with randomization of the cycles between Nunc and Falcon, 8 pregnancies were obtained in the Nunc and 10 in the Falcon dishes. The implantation rate in this second group of 50 cycles was 9 out of 61 in Falcon and 11 out of 57 in Nunc dishes.

A first prospective study of the individual straight line velocity of the spermatozoon and its influences on the fertilization rate after intracytoplasmic sperm injection.

The straight line velocity of 500 individual spermatozoa was measured immediately before their direct injection into the cytoplasm of 500 metaphase II oocytes. The straight line velocity (VSL) did not have a normal distribution (P < 0.01) and ranged between 0 and 35 microm/s. The fertilization rate (84%) was significantly (P < 0.008) higher in the quartile of the sperm population with the highest VSL compared to the fertilization rate (68%) in the quartile with the lowest VSL. Embryo cleavage and embryo quality were not different in the quartiles of the sperm population used for injection.