Clinical ICSI in the horse:

Transcription

1 Clinical ICSI in the horse: differences and similarities to human in an in vitro maturation-based system Katrin Hinrichs College of Veterinary Medicine & Biomedical Sciences Texas A&M University

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3 Standard In Vitro Fertilization Low fertilization rates, not repeatable Major barrier: Sperm fail to penetrate oocyte zona pellucida

4 Equine ICSI

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6 Horse Progesterone OV LH Luteal Follicular Luteal

7 Chorionic/ Placental hormone production Progestins

8 Chorionic/ Placental hormone production Progestins

9 Chorionic/ Placental hormone production Progestins

10 Chorionic/ Placental hormone production 2 o CLs Progestins

11 Chorionic/ Placental hormone production 2 o CLs Progestins

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13 Choi et al., 2015 CDX2 GATA6

14 Blastocyst biopsy for PGD

15 Day-7 blastocyst recovered from uterus

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17 7-d blastocyst (403 µm) After biopsy After vitrification 24 h culture Choi et al., 2011

18 Why Clinical ICSI?

19 Subfertile mare Mare cannot provide embryo for transfer Endometritis/Pyometra Uterine adhesions Cervical tear Oviductal blockage? Idiopathic

20 Post Mortem

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22 Low semen stores Only semen available is frozen, low quality or numbers Deceased stallion Aged stallion no longer fertile

23 Oocyte Recovery

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28 Recovery from Stimulated PRE-OVULATORY Follicle

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30 Management Monitor follicle growth via repeated transrectal ultrasonography Predict first day of projected responsiveness from size, shape, wall thickness Administer hcg or deslorelin Aspirate follicle 24 to 35 h later

31 Why not superovulate? Superovulation not repeatable in the horse Equine FSH can be effective - 3 follicles Ovary hard to manipulate if >1-2 large follicles present

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33 Trans-vaginal Ultrasound-guided Follicle Aspiration (TVA)

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36 Dominant Stimulated Follicle 70 85% recovery

37 24 h after hcg MI 35 h after hcg - MII Bezard et al., 1997

38 We designate these in vivo matured oocytes Culture (no hormones needed) until ~36-42 h after donor received hcg Recover at 24 h, culture 12 h Recover at 35 h, culture 1 h

39 ~ 40% blastocyst rate Jacobson et al., 2010

40 Drawbacks to Aspirating DSF: Monitoring of estrus and follicle activity in the mare Timing of gonadotropin stimulation in relation to follicle maturity Timing of aspiration at a set point after administration of gonadotropin stimulation Inability to schedule these procedures beforehand, due to variations in follicle growth Recovery at most only one maybe two oocytes Must perform ICSI at a set time after gonadotropin stimulation

41 Recovery from IMMATURE Follicles

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43 Immature Follicles: Present in the ovary throughout the year

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45 Immature Follicles: Growing follicle? Atretic follicle?

46 Work with Oocytes from Ovaries recovered Post Mortem

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48 Expanded Compact Expanded-cumulus oocytes (Ex) had a higher rate of maturation in vitro

49 Ex oocytes came from atretic follicles Oocytes from atretic follicles had a higher maturation rate

50 Developmental competence?

51 Immature Oocyte Cumulus Type and Maturation Duration (post-mortem tissue) (DMEM/F-12, 5% 0 2, 5% CO 2, 90% N 2 ) Cumulus Type Mat time n % Cleav % Blast Cp 24 h 30 h 36 h a 35 b 38 b Ex 24 h 30 h 36 h Hinrichs et al., 2005

52 Human: SN Human: NSN Monti et al., 2017

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54 LH

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59 Early results with TVA of Immature Follicles Many laboratories LOW Recovery Rate: 20% recovery rate = 2 to 6 follicles/session 1 to 2 immature oocytes /session

60 Hawley et al., 1995

61 Bovine Bovine Equine Equine Hawley et al., 1995

62 Hawley et al., 1995

63 Aspirate Scrape while emptying Fill Aspirate Scrape while emptying Fill Aspirate Scrape while emptying Fill. (6 times) Hawley et al., 1995

64 2017 Research Data TAMU 229 research transvaginal aspirations Average 71.1% oocyte recovery Average 10 oocytes per mare

65 Advantages to aspirating Immature Follicles: No hormonal stimulation needed No monitoring of estrus or follicle activity Can do on fixed schedule, e.g. once every two weeks Large number of follicles; more oocytes recovered

66 30 h maturation

67 Trials to delay onset of maturation Follicular fluid Follicle wall 6-DMAP Cycloheximide Roscovitine Butyrolactone Hinrichs et al., 1995; Alm and Hinrichs, 1996; Hinrichs et al., 2002; Franz et al., 2003

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69 Overnight holding before maturation Immature oocytes placed in modified M199 Held overnight at ROOM TEMPERATURE No detrimental effect on Maturation rate Held: 65% Control: 68% No detrimental effect on Blastocyst rate Held: 34% Control: 25% Choi et al., 2006

70 Shipped oocytes

71 Oocyte shipment to the ICSI Lab: Room Temperature vs. Cold Treatment No. oocytes No. mature oocytes/icsi (%) No. Blastocysts (% of injected) Cold (5-10 C) (23.1) a 5 (16.7) a Room Temperature (21-24 C) (37.3) b 20 (40.8) b

72 ICSI Case Load Texas A&M

73 Equal quality blastocyts?

74 Clinical ICSI results 2016 In vivo matured oocytes vs. In vitro matured (IVM) oocytes Oocyte type Blastocyst rate Pregnancy rate Ongoing pregnancy (heartbeat) In vivo 47/123 (38%) 26/36 (72%) 24/36 (67%) IVM 351/1210 (29%) 166/234 (71%) 146/234 (62%)

75 Oocyte maturation medium M199/Earle s salts 10% fetal bovine serum 5 mu/ml bovine FSH

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77 Acknowledgements Members of the Equine Embryo Laboratory Texas A&M University

78 Members of the Section of Theriogenology Texas A&M Participating ET Centers The Link Equine Research Endowment Fund, Texas A&M University