OXYGEN CONSUMPTION OOCYTE /EMBRYO QUALITY

Transcription

1 PREDICTIVE POWER OF OXYGEN CONSUMPTION IN ASSESSMENT OF OOCYTE /EMBRYO QUALITY Ana Sousa Lopes, DVM, PhD * L.I.F.E. LEUVEN INSTITUTE FOR FERTILITY AND EMBRYOLOGY * No commercial activities are related to this presentation ESHRE Campus Symposium Salzburg, Austria, 2 April 2011

2 OBJECTIVE An improved embryo selection will likely: success rates of IVF treatments Only 50% of all transferred embryos get implanted number transferred embryos by treatment cycle Multiple gestations obstetrical & neonatal complications

3 Morphology and nuclear stage Analysis culture medium Pyruvate Glucose O2 Lactate Albumin CO2 Amino acids NH3 Enzymes Early pregnancy factor PAF Oxidative stress

4 Morphology & kinetics Analysis culture medium Blastomere biopsy Gene expression Chromosomal analysis Pyruvate Glucose O2 Lactate CO2 Amino acids NH3 Enzymes Early ypregnancy factor PAF Oxidative stress

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6 WHY OXYGEN??? Oxygen provides the best indication of overall metabolic activity BECAUSE is directly related to ATP production via oxidative phosphorylation. AND. An adequate cohort of good quality mitichondria is mandatory for a GOOD quality oocyte and embryo Harvey et al. (2002)

7 MEASURING OXYGEN CONSUMPTION a) Cartesian diver techniques Fridhandler et al.1957: Mills & Brinster 1967 b) Spectrophotometric Methods Magnusson et al. 1977; Nilsson et al c) Ultramicrofluorescence techniques Houghton et al. 1996; Thompson et al d) Eletrochemical techniques Overström method Overström 1987 Scanning Electrode Technique Smith et al.1995,1999; 1999; Trimarchi et al Scanning Electrochemical microscopy Shiku et al. 2001; Koike et al. 2010

8 Nanorespirometer Embryoscope A Embryoscope C Unisense Fertilitech Non invasive Rapid Simple and practical Adapted to measure individual embryos Accurate and consistent (reproducible) Highly sensitive i Does not interfere with viability

9 THE PRINCIPLE 0.68 mm Oxygen Concentration 3 mm x 2 Min Embryo Depth C Glass Capillary Lopes et al. (2005) Reproduction

10 Oocytes (human) GV, MI (Day 0 and 1) and MII oocytes (Day 1, aged oocytes) Rates ranged between = nl/h Differences in oxygen consumption Oocytes Patients Oocytes developing in vitro vs. arrested in vitro Scott et al. (2008) RMOnline Scott et al. (2008) Proc. ESHRE Campus

11 Oocytes (human) FSH (lower oxygen consumption in patients with high FSH levels) Age (higher oxygen consumption in oocytes of younger women) Scott et al. (2008) RMOnline

12 Oocytes (human) Higher oxygen consumption of oocytes in patients stimulated with FSH Tejera et al ESHRE Rome

13 Oocytes (human) Ox xygen co onsumptio on R = p= Oxy ygen con nsumptio on R = p= Total dose of gonadotrophins Estradiol levels Tejera et al (in preparation)

14 Oocytes (human) Oxygen consumption higher in oocytes with correct fertilization Tejera et al ESHRE Rome

15 Oocytes (human) Higher oxygen consumption in oocytes producing good quality embryos (not significant) ifi Tejera et al ESHRE Rome

16 Oocytes (human) 56 couples 348 oocytes 248 embryos Day-3 1 h Tejera et al ESHRE Rome

17 Oocytes (human) Higher oxygen consumption in oocytes producing embryos that sucessfully implanted Tejera et al ESHRE Rome

18 Embryos (bovine) In vitro produced embryos (day 3: 8- cell stage) (day 7: blastocyst stage) In vivo produced embryos Lopes et al. (2005) Reproduction Lopes et al. (2007) Human Reproduction

19 Embryos (bovine) Oxy ygen co onsum mption (nl/h) n = 156 P < nl/h n = nl/h day 7 day 3 Lopes et al. (2005) Reproduction

20 Embryos (bovine) ption (nl/h) Oxy ygen co onsum n = nl/h in vitro n = nl/h in vivo Lopes et al. (2007) Human Reproduction

21 Embryos (bovine) IN VIVO EMBRYOS IN VITRO EMBRYOS ion (nl/h) consumpt Oxygen /h) Respira ation rates (nl ,10 1,80 /h) , ,20 1, , , , a Oxygen consumptio on (nl/h) a b , I II 2 III 3 4 (n =18) (n =14) (n =12) b (n =18) (n =14) (n=12) Embryo quality Morphological quality a b b c I II III IV (n = 38) (n = 52) (n = 47) (n = 19) Morphological quality Lopes et al. (2007) Human Reproduction

22 Embryos (bovine) Oxyge en consum mption (n nl/h) ion (nl/h) onsumpt Oxygen c mrna expression (G6PD) mrna expression (GLUT1) Lopes et al. (2007) Theriogenology

23 Embryos (bovine) Oxyg gen cons sumption (nl/h) a (16) a (32) a (29) a (45) female male a a (11) (11) 0 in vitro (study 1*) in vitro (study 2**) in vivo * Lopes et al. (2005) Reproduction ** Lopes et al. (2007) Human Reproduction

24 Embryos (bovine) Embryo receptacle Day 7 Day 7 2 h Lopes et al Human Reproduction

25 Embryos (bovine) OXYGEN CONSUMPTION PREGNANT NON- CATEGORY PREGNANT High ( > 1.0 nl/h) 25 %(n= 1) 75 %(n= 3) Medium ( nl/h) 100 % (n = 1 3) 0 % (n = 0) Low ( < 0.78 nl/h) 48 % (n = 11) 52 % (n = 12) Lopes et al. (2007) Human Reproduction

26 Embryos (human) tion nsumpt l/sec) ygen co (fmo Oxy n = P < 0.05 embryos that implanted n = embryos that did not implanted Tejera et al. (2010) Fertility & Sterility

27 Embryos (human) Scanning Electrochemical Microscopy (SECM) Koike et al. (2010) Fertility and Sterility

28 Embryoscope TM Unisense Repetitive measurements with acquisition of digital images from each embryo

29 Embryos (bovine) Denuded zygotes (previous IVF for 6 h) Loaded in the Embryoslide Repetitive O2 measurements + Digital pictures

30 Embryos (bovine) 0,6 (nl/h) Mean resp piration rate 0,58 0,56 0,54 0,52 0,5 0,48 0, Time after fertilization (h) non-fertilized in vitro-matured oocytes in vitro-produced zygotes Lopes et al. (2010) Human Reproduction

31 Embryos (bovine) Mea an respiration rate (nl/h) 0,59 0,57 0,55 0,53 0,51 0,49 0, Time after fertiliztion (h) a) b) Mean respiration rate (nl/h) 0,59 0,57 0,55 0, ,51 0, ,56 Time after fertilization (h) respiration rate (n nl/h) 0,58 0,56 0,54 0,52 l/h) espiration rate (nl 0,54 0,52 0,5 Mean 0,5 Mean r 0,48 0,48 0, Time after fertilization (h) c) d) Time after fertilization (h) Lopes et al. (2010) Human Reproduction

32 Embryos (bovine) Time in relation to Mean oxygen SEM cleavage (min) cons. (nl/h) a b,c b,d b c,d Lopes et al. (2010) Human Reproduction

33 Embryos (bovine) Callesen et al Depart. Genetics & Biotechnology, Aahrus University, Denmark

34 Embryos (bovine)

35 Embryos (bovine) 114 embryos Callesen et al Genetics & Biotechnology Aahrus University, Denmark

36 Embryos (murine) Cleavage stage (2-8cell) individual oxygen consumption ranges between nl/h Oxygen consumption rises slightly at the morula stage and significantly at the expanded blastocys stage Ottosen et al J Assist Reprod Genet

37 Embryos (murine) ONLY at the Morula stage was higher oxygen consumption associated with subsequent development to the blastocysts stage. Ottosen et al J Assist Reprod Genet

38 Embryos (human) Different O2 rates and profiles for embryos developing vs. arresting in vitro Non used thawed embryos (2PN to blastocysts) 1 & 3PN embryos Day 2 and 3 discarded embryos Day 4 abnormal embryos from PGD Scott et al. (2008) Proc. ESHRE Campus

39 Embryos (human) Oxygen consumption is steady until day 4, ranging bt nl/h Oxygen consumption rises is consistent with blastocyst formation (range nl/h) Scott et al. (2008) Proc. ESHRE Campus

40 THUS.. Oxygen consumption measured once/continuously is a valuable parameter for assessing oocyte/embryo metabolism Combining oxygen consumption with other viability criteria may improve in the selection of superior embryos before transfer. Patterns of oxygen consumption around the time of cleavage may also help in selecting better quality embryos g y p g q y y Analysis of metabolism (and thus oxygen) can be crucial for implementation of the SET policies (mostly embryos similar morphology). )

41 Henrik Callesen TRANS EMBRYO GENETICS Denmark Søren Madsen Torben Greve Marcus Messeguer AlbertoTejera Jeremy Thompson Michelle Lane Niels Ramsing