In-vitro maturation of immature human oocytes
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1 In-vitro maturation of immature human oocytes Focusing on the methodology Byron Asimakopoulos, PhD Associate Professor Faculty of Medicine, Democritus University of Thrace
2 In-vitro maturation The maturation in culture of immature oocytes after their recovery from follicles that may or may not have been exposed to exogenous gonadotropins. Prophase I Metaphase II Metaphase I Mataphase II
3 History of IVM 1935: Pincus & Enzmann reported that immature rabbit oocytes were capable of spontaneous maturation and fertilization in-vitro. [Pincus G, Enzmann EV (1935). J Exp Med, 62(5): ] 1965: Edwards RG. (1965a): Maturation in vitro of mouse, sheep, cow, pig, rhesus monkey and human ovarian oocytes. Nature,208: Edwards RG (1965b): Maturation in vitro of human ovarian oocytes. Lancet, II: : Cha et al reported the first birth using IVM oocytes from an oocyte donor program [Cha KY, Koo JJ, Ko JJ, Choi DH, Han SY, Yoon TK (1991). Fertil Steril, 55: ] 1994: Trounson et al reported the first birth using IVM oocytes from an unstimulated PCO patient [Trounson A, Wood C, Kausche A (1994). Fertil Steril, 62(2): ]
4 Clinical applications of IVM Patients at risk of OHSS PCOS patients Patients with a history of OHSS Patients with limited time for controlled ovarian stimulation Patients who need initiation of gonadotoxic treatments Patients at risk of sustained elevations of E2 Patients with hormonally sensitive tumors
5 IVM in order to rescue immature oocytes retrieved in standard IVF cycles IVM in unstimulated or mildly stimulated cycles
6 The immature oocytes collected in standard IVF cycles are inherently abnormal They have failed to respond to ovulatory gonadotropin stimulus They show grossly aberrant gene expression (2766 genes) They have limited developmental competence after IVF/ICSI Jones GM, Cram DS, Song B, Magli MC, Gianaroli L, Lacham-Kaplan O, et al. Gene expression profiling of human oocytes following in vivo or in vitro maturation. Hum Reprod 2008;23: Nogueira D, Staessen C, Van de Velde H, Van Steirteghem A. Nuclear status and cytogenetics ofembryos derived from in vitro-matured oocytes. Fertil Steril 2000;74: Strassburger D, Goldstein A, Friedler S, Raziel A, Kasterstein E, Mashevich M, et al. The cytogenetic constitution of embryos derived from immature (metaphase I) oocytes obtained after ovarian hyperstimulation. Fertil Steril 2010;94:971 8.
7 Virant-Klun et al (2013). BioMed Research International
8 To prime or not to prime? No stimulation (natural cycle) FSH for 3-6 days, retrieval on day IU of hcg prior to retrieval Combination of FSH and hcg
9 The immature oocytes in the lab Culture in a conventional IVF medium? Culture in specific maturation media?
10 The maturation media have been designed for immature cumulus oocyte complexes Oocyte IVM media are intended for the culture of intact cumulus-oocyte complexes (COC). As COC contain cumulus cells, but just one oocyte, IVM media are designed principally to meet the nutritional needs of the somatic cells. Hence, IVM media are complex media capable of supporting the high metabolic requirements of the COC (e.g., very high glycolytic activity), whereas cleavage media are designed for the low metabolic needs of the early embryo (e.g., utilize glucose poorly) Gilchrist et al (2011). Fertil Steril, 96(2): e141
11 Sutton ML, Gilchrist RB, Thompson JG (2003). Hum Reprod Update, 9(1): 35-48
12 Factors promoting IVM Maturation Promoting Factor (MPF) Mitogen Activated Protein Kinase (MAPK) Oocyte size (diameter >115μ) FSH LH (few LHR in cumulus cells no (?) LHR in oocyte) Epidermal Growth Factor (EGF) Meiosis Activating Sterol (FF-MAS) Growth Hormone (GH) Insulin Insulin-like Growth Factor-I (IGF-I) Prolactin
13 The usual procedure Two types of media Washing and Preincubation of immature oocytes in the first medium Incubation for >28 hours in the second medium (maturation medium) The maturation medium is usually supplemented with FSH, hcg and serum proteins
14 Supplementation with FSH/LH SAGE IVM system
15 Supplementation with FSH/LH SAGE IVM system
16 IVM for how long?
17 Oocytes obtained after oophorectomy Gumala S, Kaufman ML, Racowsky C (1993). Assis Reprod Technol Androl, 4: 1-14
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19 Common questions Maturation rate? Fertilization: Conventional IVF or ICSI? Embryo development: Do the IVM oocytes have the same developmental competence as the in-vivo matured oocytes? Implantation rate? Pregnancy rate?
20
21 Denuded oocytes
22 Preliminary results on the efficacy of a new IVM system N= 174 immature oocytes AB1a: GV oocytes for 4 hours AB1b: GV & MI oocytes for 46 hours Glucose, essential & non-essential aminoacids, sodium pyrouvate, sodium bicarbonate & gonadotropins maturation % GV MI GV MII MI MII
23 Álvarez et al (2013). Indian J Med Res, 137:
24 Strowitzki T (2013) Arch Gynecol Obstet, 288: Roesner et al (2012) Acta Obstet Gynecol Scand, 91: 22-27
25
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27 Modified Tyrode s solution supplemented with human follicular fluid and amino acids
28
29 Vanhoutte et al (2005). Reprod Biol Endocrinol, 3(71). Denuded oocytes
30 Clinical outcome: IVF vs. IVM No randomized, controlled trials currently exist comparing IVF & IVM
31 Conclusions IVM is a useful method especially for patients at risk for OHSS, patients with PCOS or PCO-like ovaries, patients with estrogen sensitive tumors or patients with a limited time prior to gonadotoxin treatments Priming with HCG or/and FSH appears to improve pregnancy & implantation rates IVM with FSH/LH COCs Fertilization is possible either with conventional IVF or ICSI Pregnancy and implantation rates are lower than COH/IVF ~2500 children have been conceived with IVM, therefore the safety of IVM with regard to malformation and development outcomes can not be assessed accurately
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