Triggering final oocyte maturation with a GnRH agonist does not affect apoptosis of follicular granulosa-lutein cells

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We have shown that a low level of apoptosis of human granulosa-lutein cells (GL cells) from IVF cycles is associated with younger age and pregnancy. We would therefore expect a low level of apoptosis in egg donors. The present study was designed to test the effect of different ovulation induction protocols (antagonist/hCG, antagonist/GnRH agonist, downregulation/hCG) on in vitro GL cells apoptosis in young donors. We were particularly interested in whether administration of a GnRH agonist to trigger final oocyte maturation instead of hCG in the antagonist group would increase the rate of in vitro apoptosis of gonadotropin-exposed GL cells. In vitro assays. 19 egg donors with a mean age of 22.42 ± 2.87 underwent ovulation induction with recombinant FSH combined with recombinant LH or HMG using a downregulation or an antagonist protocol. The ovarian response was monitored by transvaginal ultrasound and blood estradiol levels as needed. Final oocyte maturation was triggered by either hCG or leuprolide acetate and the egg retrieval was performed 35.5 hours later. GL cells were collected from pooled follicular fluid after oocyte recovery. Cells were washed and further purified using a 50% percoll gradient and anti-CD45 coated magnetic beads. After 48 hour culture without serum the percentage of apoptosis was determined under a fluorescent microscope after staining with 10 μM caspACE FITC-VAD-FMK (a fluorescent marker for activated caspases) and 0.05 mg/ml propidium iodide. Statistical analysis was carried out using the Student's t-test. Overall the apoptosis rate was low, as expected for a group of young donors (19.06% ± 4.01, n = 19). There were no significant differences in the percentage of apoptosis among the three groups of egg donors analyzed (antagonist/hCG: 17.35 ± 4.64, n = 5; antagonist/GnRH agonist 19.55 ± 2.61, n = 5; downregulation/hCG: 19.73 ± 4.42, n = 9; P>0.05). Moreover, the level of apoptosis observed in the donors was significantly lower than that observed in a group of infertility patients with a mean age of 32.47 ± 3.22 undergoing IVF during the same period of time (19.06% ± 4.01, n = 19 vs. 24.92 ± 4.25, n = 19; P<0.05). Triggering of ovulation with a GnRH agonist does not increase apoptosis of gonadotrophin exposed granulosa-lutein cells. Furthermore, there is no difference between downregulation and antagonist protocols.