1. mRNA EXPRESSION OF APOPTOTIC MARKERS AND ANTIOXIDANT ENZYMES IN CUMULUS AND GRANUOLSE CELLS FROM PERIOVULATORY FOLLICLES OF YOUNG WOMEN WITH LOW OVARIAN RESERVE R. NUÑEZ-CALONGE 1, S.CORTES 1, L.RANCAN 2, E.VARA 2, L.ORTEGA 1, P.CABALLERO 1, J.FERNANDEZ- TRESGUERRES 2. 1 Clínica Tambre, Madrid, SPAIN 2 Dpto. Fisiología, Universidad Complutense Madrid

2. Introduction

4. The antioxidant GST has been found to be decreased in patients with low response to COS. MDA, as one of the most important markers of OS, as well as the proinflammatory cytoquine IL- 6, have been found to be increase in patients with reduced ovarian reserve.

5. Introduction

6. Hypothesis An altered response to gonadotropin stimulation in women with infertility may be associated with altered miRNA expression in somatic follicular cells. We therefore sought to determine whether expression of miRNAs in cumulus and granulose cells is altered in women with infertility who demonstrates poor response to COH-IVF.

7. Objective To investigate apoptosis and oxidative stress in granulose (GC) and cumulus cells (CC) from periovulatory follicles of young women with low response to COH. To this end, we compared the levels of mRNA coding for antioxidant enzymes and apoptotic markers in women with low response in in vitro fertilization (IVF) and fertile oocyte donors as controls.

8. Methods 20 healthy, fertile, Donors (< 35 years old) 15 low responders Patients (< 35 years old) COS rFSH+ antagonist GnRH Patients enrolled in this study were selected on the basis of their ovarian response (with normal FSH and/or AMH levels) and only patients with at least a previous cycle with low response were included.

9. 2.900 rpm, 10´ Methods: Isolation of Granulose cells The supernatant was discarded, and the cell pellet was resuspended in DMEM/Ham’s F12. GCs were separated from erythrocytes on a 40% Percoll gradient. GCs were concentrated by centrifugation, and the cell pellet was resuspended in fresh medium.

10. Methods: Isolation of Cumulus cells Retrieved cumulus-oocyte complexes were placed in culture medium (G-MOPS, VitroLife), and cumulus cells were dissected from the oocyte mechanically in the absence of hyaluronidase

11. Methods  Superoxide dismutase (SOD)  Catalase Antioxidant enzymes  Caspase 3 and Caspase 9  Bcl-2 Apoptotic markers  BAX We carried out RT-PCR analysis on CGs and CCs from pooled follicular aspirates of each pacient:

12. Methods Statistical analysis was performed using one-way analysis of variance (ANOVA), followed by the unpaired Student’s t-test. To evaluate mRNA levels of SOD,catalase, caspase 3, caspase 9, Bax and Bcl2 in donors and patients, we carried out RT–PCR analysis on GCs and CCs from pooled follicular aspirates of each patient. The reverse transcription of RNA was performed using the RETROscript® Reverse Transcription Kit (Ambion, Life Technologies, Texas, USA) following the manufacturer’s instructions

13. Results

14. Mean ± SD DonorsPatients P value Catalase 1,47±0,110,87±0,06.005 SOD 1,55±0,071,42±0,02 NS Caspase 3 0,34 ±0,010,71±0,049.0006 Caspase 9 0,81±0,061,37±0,1.009 BAX 1,19±0,022,11±0,2.0018 Bcl2 1,02±0,021,46±0,09.006 Levels of mRNA coding for antioxidant enzymes and apoptotic markers in GCs of low responders patients compared with controls.

15. Results Mean ± SD DonorsPatients P value Catalase 1,66±0,140,99±0,06.0037 SOD 1,19±0,071,2±0,1 NS Caspase 3 1,008 ±0,121,01±0,006 NS Caspase 9 1,05±0,061,1±0,07 NS BAX 1,26±0,062,95±0,19 0.08 Bcl2 1,35±0,051,37±0,03NS Levels of mRNA coding for antioxidant enzymes and apoptotic markers in CCs of low responders patients compared with controls.

16. Results p<0.01 Catalase mRNA expression levels in cumulus and granulosa cells from donors and patients. Values are means ± SEM p<0.01

17. Results p<0.01 Caspase 3 and Caspase 9 mRNA expression levels in granulose cells from donors and patients. Values are means ± SEM

18. Results p<0.01 BAX mRNA expression levels in cumulus and granulosa cells from donors and patients. Values are means ± SEM p<0.01

19. Conclusions Our results suggest a lower antioxidant capacity and an increased apoptosis level in CC and GC of women with low response to COH compared with fertile oocyte donors. Otherwise, mRNAs coding for antioxidant enzymes and apoptotic markers are differentially expressed in granulose and cumulus cells. In addition, in this study, we demonstrate a significant difference between women with poor and normal responses to COH and we found there were a good correlation between increased GCs oxidative stress, apoptosis and low ovarian response, suggesting that oxidative stress could influence oocyte production.

20. Conclusions The relations of some antioxidant enzymes like SOD, and apoptotic markers in CC with OE and its implication in ovarian reserve needs to be addressed in further studies on a larger sample. Identifying optimal biomarkers that reliably predict ovarian response in the clinical setting could lead to developing antioxidant therapies for these poorer prognosis women undergoing IVF treatment.

21. THANK YOU FOR YOUR ATTENTION