Josiane Van der Elst

Follicular growth in fresh and cryopreserved human ovarian cortical grafts transplanted to immunodeficient mice

OBJECTIVEnTo investigate follicle growth in fresh and cryopreserved human ovarian cortical grafts transplanted to immunodeficient mice.nnnSTUDY DESIGNnFresh or frozen-thawed human ovarian cortex was grafted subcutaneously or under the kidney capsule of 43 mice (35 nude mice and eight SCID mice), 14 of which were non-stimulated controls, 21 injected intra-peritoneally with gonadotrophins during 2 weeks and eight injected during 3 months. Follicle count was compared by Chi-square.nnnRESULTSnProportions of primordial follicles were significantly lower in grafts than in the tissue before transplantation in gonadotrophin-stimulated mice (37% versus 79%), but not in non-stimulated mice (51% versus 74%). Proportions of primary and secondary follicles were increased after transplantation indicating early follicular growth. One antral follicle was observed in a graft in a mouse stimulated for 3 months.nnnCONCLUSIONnPrimordial follicles in fresh or frozen-thawed human ovarian cortex transplanted under the kidney capsule or subcutaneously can grow and are responsive to hormonal stimulation.nnnCONDENSATIONnPrimordial follicles in fresh and cryopreserved human ovarian cortical grafts can initiate growth after transplantation to immunodeficient mice

Maturation of Mouse Primordial Follicles by Combination of Graftingand In Vitro Culture

Abstract Cryopreservation of ovarian cortical tissue and subsequent transplantation or in vitro culture of follicles are technologies under development with the aim to safeguard fertility in patients with gonadal failure. In the present study, we investigated whether primordial follicles could be triggered to full maturation by a combination of in vivo transplantation and in vitro culture in a mouse model. In a first step, newborn mouse ovaries containing only primordial follicles were allotransplanted under the renal capsule of ovariectomized recipient mice. The second step was to mechanically isolate growing preantral follicles from the graft and culture these in vitro to maturity. In our experiment, one newborn mouse ovary was transplanted under the renal capsule of each 8- to 12-wk-old F1 (C57Bl/6j × CBA/Ca) female ovariectomized recipient (n = 26). Two weeks after transplantation, all 26 grafts were recovered. Four grafts were processed for histology and showed that developmental stages of follicles in 14-day-old ovarian grafts were comparable to those in 14-day-old mouse ovaries. The 22 remaining grafts were used for mechanical isolation of preantral follicles. As a control group, preantral follicles isolated from ovaries of 14-day-old mice were used. The mean preantral follicle yield per ovary was 11 in the transplant group versus 33 in the control group. Follicles were cultured individually in 20-μl droplets of α-MEM supplemented with 100 mIU rFSH and 5% fetal bovine serum for 12 days under an atmosphere of 5% CO2 in air at 37°C. By Day 12 of culture, 66.5% of follicles retained their oocytes in the grafting group versus 97.5% in the control group (P < 0.001). Final oocyte maturation was induced by addition of 2.5 IU/ml hCG. At 14–16 h post-HCG, the percentages of oocytes showing germinal vesicle breakdown and polar body extrusion were significantly higher in the control group (90.6% and 82.8%) compared to the grafting group (60% and 45%). The mean diameter of the mature oocytes of the grafting group (69.9 ± 4.45 μm) was similar to that of oocytes from the control group (70.5 ± 2.35 μm). Our results suggest that maturation of mouse primordial follicles is feasible by combination of in vivo transplantation and in vitro culture. This two-step strategy may be an attractive model for promoting the growth and maturation of primordial follicles from other species.

Clinical benefit of metaphase I oocytes.

BackgroundWe studied the benefit of using in vitro matured metaphase I (MI) oocytes for ICSI in patients with a maximum of 6 mature metaphase II (MII) oocytes at retrieval.MethodsIn 2004, 187 ICSI cycles were selected in which maximum 6 MII oocytes and at least one MI oocyte were retrieved. MI oocytes were put in culture to mature until the moment of ICSI, which was performed between 2 to 11 hours after oocyte retrieval (day 0). In exceptional cases, when the patient did not have any mature oocyte at the scheduled time of ICSI, MI oocytes were left to mature overnight and were injected between 19 to 26 hours after retrieval (day 1). Embryos from MI oocytes were chosen for transfer only when no other good quality embryos from MII oocytes were available. Outcome parameters were time period of in vitro maturation (IVM), IVM and fertilization rates, embryo development, clinical pregnancy rates, implantation rates and total MI oocyte utilization rate.ResultsThe overall IVM rate was 43%. IVM oocytes had lower fertilization rates compared to in vivo matured sibling oocytes (52% versus 68%, P < 0.05). The proportion of poor quality embryos was significantly higher in IVM derived oocytes. One pregnancy and live birth was obtained out of 13 transfers of embryos exclusively derived from IVM oocytes. This baby originated from an oocyte that was injected after 22 hrs of IVM.ConclusionFertilization of in vitro matured MI oocytes can result in normal embryos and pregnancy, making IVM worthwhile, particularly when few MII oocytes are obtained at retrieval.

Long term effects of micro-surgical testicular sperm extraction on androgen status in patients with non obstructive azoospermia

BackgroundThe aim of our study was to review the results of microsurgically performed testicular sperm extraction (TESE) and to evaluate its possible long term effects on serum testosterone (T).MethodsWe operated on 48 men (35 +/- 8 years) with non-obstructive azoospermia (NOA). If no spermatozoa were found following a micro epididymal sperm extraction (Silber et al., 1994) and testicular biopsy, testicular microdissection was performed or multiple microsurgical testicular biopsies were taken. The mean follow-up of the serum T was 2.4 +/- 1.1 years.ResultsSperm was retrieved in 17/48 (35%) of the men. The per couple take home baby rate if sperm was retrieved was 4/17 (24%). Serum T decreased significantly at follow-up (p < 0.05) and 5/31 (16%) de novo androgen deficiencies developedConclusionIn patients with non-obstructive azoospermia in whom no spermatozoa were found following a micro epididymal sperm aspiration and a simple testicular biopsy, we were able to retrieve spermatozoa in 35% of the men. The take home baby rate was 24% among couples with spermatozoa present upon TESE. De novo androgen deficiency occurred in 16% of the male patients following TESE indicating that, in men with NOA, long term hormonal follow up is recommended after TESE.

Effect of ionomycin on oocyte activation and embryo development in mouse

Artificial oocyte activation using the calcium ionophore ionomycin is applied successfully in assisted reproduction but some concern exists on the clinical use. The aims of the present study were to optimize the oocyte activation scheme and to address embryo toxicity in a mouse model. Efficiency of oocyte activation and subsequent development was evaluated and ionomycin was found to be an efficient activator at 10 micromol/l. An improved effect of a second exposure to 5 micromol/l ionomycin on blastocyst development was observed. Toxicity of ionomycin on embryos was then investigated by evaluating pre- and post-implantation development of in-vivo fertilized oocytes following exposure to ionomycin. Blastocyst development, blastocyst cell numbers in trophectoderm and inner cell mass were not different between treated and non-treated zygotes. Also implantation rates and fetal parameters such as length, weight and morphological parameters were similar between the fetuses originating from zygotes treated with ionomycin and non-treated zygotes. Furthermore, healthy offspring originating from ionomycin-treated zygotes was born. In conclusion, no adverse effects of ionomycin on in-vitro or in-vivo mouse embryo development were noticed, giving arguments in favour of the use of ionomycin, although negative long-term effects of this compound cannot be excluded at present.

Fertilization of Mouse Oocytes from In Vitro-Matured Preantral Follicles Using Classical In Vitro Fertilization or Intracytoplasmic Sperm Injection

Abstract Early preantral mouse follicles with a diameter of 110–160 μm were cultured in vitro for 10 or 12 days. Mature oocytes were retrieved following hCG, and fertilization was attempted either by in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI). Two-cell and blastocyst formation rates and blastocyst cell numbers were compared between 10-day and 12-day in vitro-matured oocytes versus in vivo-matured oocytes. Uncleaved IVF oocytes were subjected to chromosome analysis. The 2-cell formation rate was significantly improved by ICSI compared with IVF both in 10-day (72.1% versus 56.1%; P = 0.03) and 12-day cultures (74.1% versus 54.5%; P = 0.028). Cytogenetic analysis of uncleaved MII oocytes following IVF showed that about 30% of MII oocytes showed no sign of sperm penetration. The blastocyst formation rate was significantly lower in 12-day versus 10-day cultures, whether fertilization was by IVF (40.7% versus 62.4%, P = 0.016) or by ICSI (32.5% versus 57.1%, P = 0.035). Blastocyst cell numbers from IVF and ICSI 10-day groups were similar and both significantly higher (P < 0.001) than from IVF 12-day cultures. All above expressed values were significantly higher for in vivo-matured oocytes. In conclusion, fertilization of oocytes from in vitro-matured mouse preantral follicles can be optimized with ICSI, giving significantly higher 2-cell formation rates than IVF. Blastocyst formation rate was not influenced by the technique of fertilization but rather by the extent of the in vitro culture period. Best results on preimplantation development of oocytes for in vitro-matured preantral follicles were obtained with ICSI on oocytes from 10-day in vitro cultures.

In Vitro Parthenogenetic Development of Mouse Oocytes Following Reciprocal Transfer of the Chromosome Spindle Between In Vivo-Matured Oocytes and In Vitro-Matured Oocytes

Abstract Mouse follicles grown in vitro from preantral to mature stages yield oocytes that can be fertilized in vitro, but embryonic development is poor. To investigate whether this poor development is due to a nuclear or a cytoplasmatic factor, we designed an experiment in which the MII chromosome spindle was exchanged between in vitro-matured oocytes and in vivo-matured oocytes by electrofusion. Subsequent embryo development was evaluated by blastocyst formation rate and blastocyst cell number after parthenogenetic activation. Electrofusion was successful in 62–78% of the oocytes. Transfer of the spindle apparatus from in vitro-matured oocytes to the in vivo MII cytoplasmic environment resulted in a high rate of blastocyst development, whereas in the reverse situation (transfer of the nucleus from in vivo-matured oocytes into in vitro-matured MII cytoplasm) poor quality embryos and a low rate of blastocyst formation was observed. These results indicate that the low developmental competence of in vitro-matured oocytes from mouse preantral follicles after activation is caused by the cytoplasmic component rather than the nuclear component.

Timing of FSH-stimulation and follicular development in cryopreserved human ovarian grafts

Cryopreserved human primordial follicles grafted into immunodeficient hosts are susceptible to gonadotrophic stimulation but the optimal interval between grafting and stimulation has not been determined. The effect of stimulation with FSH at different time intervals after grafting was therefore investigated. Cryopreserved human ovarian cortical grafts from an androgen pre-treated female-to-male transsexual patient were transplanted into four groups of four non-obese, diabetic severe combined immune deficiency (NOD-SCID) mice. Ten, 12, 14 and 17 weeks after grafting, stimulation was started with daily intra-peritoneal injections of 5 IU of recombinant FSH for 14 days. Grafts were recovered and serially sectioned for counting the number of follicles and determining the different stages of development. Significantly more primary and secondary follicles were found in all stimulated groups compared with the non-stimulated control. This progression from the primordial to growing primary and secondary stages was most significant in the 14 weeks interval group. Formation of antral follicles was scarce and was only noticed in the 10 and 17 week groups. Cystic follicles and follicles with structural oocyte abnormalities were encountered in each group. Whether this phenomenon is related to the hormonal state of the tissue donor or to the cryopreservation and transplantation procedures is the subject of further investigation. It is concluded that the interval between grafting of ovarian tissue and the start of gonadotrophic stimulation may be important for an optimal follicular development after grafting.

Biological aspects of testicular sperm extraction.

Since the first reports of successful pregnancies after treatment with intra cytoplasmic sperm injection (ICSI) in humans, intensive investigations focused on the use of testicular spermatozoa and immature sperm cells in the treatment of azoopsermic patients. Several studies explore the technical development of the preparation, isolation and cryo storage of testicular germ cells. Other studies focus on ICSI itself and try to identify the biochemical and biophysical processes involved in fertilisation after injection of a testicular sperm cell into a human oocyte. Indications for azoospermic patients to whom this first line treatment can be offered are becoming more defined. But one of the major concerns is of course the safety of the technique, especially, for the health and reproductive life of the babies born after application of ICSI with testicular germ cells. An evaluation of ICSI with testicular germ cells is presented in this manuscript.

Culture Medium Preferences of Pre-Implantation Cloned Mouse Embryos

Somatic cloning technology involves the transfer of a somatic cell nucleus into an enucleated oocyte, followed by activation and in vitro culture. Efficiency in terms of live offspring generally remains very low. Little attention has been devoted so far to the impact of culture environment on cloned embryo development. Failure of genomic reprogramming of the donor nucleus in nuclear transfer (NT) experiments could lead to an altered phenotype in these cloned embryos that could be manifested by different medium preferences of the NT embryos. We describe here the application of sequential culture media to support preimplantation development of mouse embryos reconstructed using conventional NT techniques. Embryo-quality analysis was performed on NT blastocysts obtained. Additionally, NT embryos that arrested during development also were analyzed.