Fine morphological assessment of quality of human mature oocytes after slow freezing or vitrification with a closed device: a comparative analysis

Reproductive Biology and Endocrinology

Stefania A Nottola²  Giuseppe Familiari²  Selenia Miglietta²  Sandra Cecconi³  Michela Lappi¹  Andrea Borini¹  Guido Macchiarelli³  Veronica Bianchi¹ 

[1] Casa di Cura Città di Udine, Udine, Italy, affiliated to Tecnobios Procreazione, Centre for Reproductive Health, Bologna, Italy;Department of Anatomy, Histology, Forensic Medicine and Orthopaedics, La Sapienza University, Rome, Italy;Department of Life, Health and Environmental Sciences, University of L´Aquila, L’Aquila, Italy

关键词: Human;    Ultrastructure;    Vitrification;    Slow freezing;    Cryopreservation;    Oocyte;   

Others  :  1139728
DOI  :  10.1186/1477-7827-12-110

 received in 2014-07-03, accepted in 2014-10-14,  发布年份 2014

【 摘 要 】

Background

Human mature oocytes are very susceptible to cryodamage. Several reports demonstrated that vitrification might preserve oocyte better than slow freezing. However, this is still controversial. Thus, larger clinical, biological and experimental trials to confirm this concept are necessary. The aim of the study was to evaluate and compare fine morphological features in human mature oocytes cryopreserved with either slow freezing or vitrification.

Methods

We used 47 supernumerary human mature (metaphase II) oocytes donated by consenting patients, aged 27-32 years, enrolled in an IVF program. Thirtyfive oocytes were cryopreserved using slow freezing with 1.5 M propanediol +0.2 M sucrose concentration (20 oocytes) or a closed vitrification system (CryoTip Irvine Scientific CA) (15 oocytes). Twelve fresh oocytes were used as controls. All samples were prepared for light and transmission electron microscopy evaluation.

Results

Control, slow frozen/thawed and vitrified/warmed oocytes (CO, SFO and VO, respectively) were rounded, 90–100 μm in diameter, with normal ooplasm showing uniform distribution of organelles. Mitochondria-smooth endoplasmic reticulum (M-SER) aggregates and small mitochondria-vesicle (MV) complexes were the most numerous structures found in all CO, SFO and VO cultured for 3–4 hours. M-SER aggregates decreased, and large MV complexes increased in those SFO and VO maintained in culture for a prolonged period of time (8–9 hours). A slight to moderate vacuolization was present in the cytoplasm of SFO. Only a slight vacuolization was present in VO, whereas vacuoles were almost completely absent in CO. Amount and density of cortical granules (CG) appeared abnormally reduced in SFO and VO, irrespective of the protocol applied.

Conclusions

Even though, both slow freezing and vitrification ensured a good overall preservation of the oocyte, we found that: 1) prolonged culture activates an intracellular membrane “recycling” that causes the abnormal transformation of the membranes of the small MV complexes and of SER into larger rounded vesicles; 2) vacuolization appears as a recurrent form of cell damage during slow freezing and, at a lesser extent, during vitrification using a closed device; 3) premature CG exocytosis was present in both SFO and VO and may cause zona pellucida hardening.

【 授权许可】

   
2014 Bianchi et al.; licensee BioMed Central Ltd.

【 图 表 】

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【 参考文献 】

• [1]Practice Committees of American Society for Reproductive Medicine; Society for Assisted Reproductive Technology: Mature oocyte cryopreservation: a guideline. Fertil Steril 2013, 99:37-43.
• [2]Chen C: Pregnancy after human oocyte cryopreservation. Lancet 1986, 1:884-886.
• [3]Jackowski S, Leibo SP, Maxur P: Glycerol permeabilities of fertilized and infertilized mouse ova. J Exp Zool 1980, 212:329-241.
• [4]Leibo SP: Water permeability and its activation energy of fertilized and unfertilized mouse ova. J Membr Biol 1980, 53:179-188.
• [5]Coticchio G, Bonu MA, Sciajno R, Sereni E, Bianchi V, Borini A: Truths and myths of oocyte sensitivity to controlled rate freezing. Reprod Biomed Online 2007, 15:24-30.
• [6]Gook DA, Edgar DH: Human oocyte cryopreservation. Hum Reprod Update 2007, 13:591-605.
• [7]Edgar DH, Gook DA: A critical appraisal of cryopreservation (slow cooling versus vitrification) of human oocytes and embryos. Hum Reprod Update 2012, 18:536-554.
• [8]Matson PL, Graefling J, Junk SM, Yovich JL, Edirisinghe WR: Cryopreservation of oocytes and embryos: use of a mouse model to investigate effects upon zona hardness and formulate treatment strategies in an in-vitro fertilization programme. Hum Reprod 1997, 12:1550-1553.
• [9]Porcu E, Fabbri R, Seracchioli R, Ciotti PM, Magrini O, Flamigni C: Birth of a healthy female after intracytoplasmic sperm injection of cryopreserved human oocytes. Fertil Steril 1997, 68:724-726.
• [10]Asghar W, El Assal R, Shafiee H, Anchan RM, Demirci U: Preserving human cells for regenerative, reproductive, and transfusion medicine. Biotechnol J 2014, 9:895-903.
• [11]Bianchi V, Lappi M, Bonu MA, Borini A: Oocyte slow freezing using a 0.2-0.3 M sucrose concentration protocol: is it really the time to trash the cryopreservation machine? Fertil Steril 2012, 97:1101-1107.
• [12]Levi Setti PE, Porcu E, Patrizio P, Vigiliano V, de Luca R, d'Aloja P, Spoletini R, Scaravelli G: Human oocyte cryopreservation with slow freezing versus vitrification. Results from the National Italian Registry data, 2007–2011. Fertil Steril 2014, 102:90-95.e2.
• [13]Martínez-Burgos M, Herrero L, Megías D, Salvanes R, Montoya MC, Cobo AC, Garcia-Velasco JA: Vitrification versus slow freezing of oocytes: effects on morphologic appearance, meiotic spindle configuration, and DNA damage. Fertil Steril 2011, 95:374-377.
• [14]Arav A, Natan Y: Vitrification of oocytes: from basic science to clinical application. Adv Exp Med Biol 2013, 761:69-83.
• [15]Paffoni A, Guarneri C, Ferrari S, Restelli L, Nicolosi AE, Scarduelli C, Ragni G: Effects of two vitrification protocols on the developmental potential of human mature oocytes. Reprod Biomed Online 2011, 22:292-298.
• [16]Parmegiani L, Cognigni GE, Filicori M: Vitrification carriers and European regulation. Fertil Steril 2012, 97:e24. author reply e25
• [17]Valbuena D, Póo ME, Aguilar-Gallardo C, Martinez S, Cobo AC, Pellicer A, Simón C: Comparison of Cryotip vs. Cryotop for mouse and human blastomere vitrification. Fertil Steril 2012, 97:209-217.
• [18]Papatheodorou A, Vanderzwalmen P, Panagiotidis Y, Prapas N, Zikopoulos K, Georgiou I, Prapas Y: Open versus closed oocyte vitrification system: a prospective randomized sibling-oocyte study. Reprod Biomed Online 2013, 26:595-602.
• [19]Bonetti A, Cervi M, Tomei F, Marchini M, Ortolani F, Manno M: Ultrastructural evaluation of human metaphase II oocytes after vitrification: closed versus open devices. Fertil Steril 2011, 95:928-935.
• [20]Seet VY, Al-Samerria S, Wong J, Stanger J, Yovich JL, Almahbobi G: Optimising vitrification of human oocytes using multiple cryoprotectants and morphological and functional assessment. Reprod Fertil Dev 2013, 25:918-926.
• [21]Nottola SA, Macchiarelli G, Coticchio G, Bianchi S, Cecconi S, De Santis L, Scaravelli G, Flamigni C, Borini A: Ultrastructure of human mature oocytes after slow cooling cryopreservation using different sucrose concentrations. Hum Reprod 2007, 22:1123-1133.
• [22]Nottola SA, Coticchio G, De Santis L, Macchiarelli G, Maione M, Bianchi S, Iaccarino M, Flamigni C, Borini A: Ultrastructure of human mature oocytes after slow cooling cryopreservation with ethylene glycol. Reprod Biomed Online 2008, 17:368-377.
• [23]Nottola SA, Coticchio G, Sciajno R, Gambardella A, Maione M, Scaravelli G, Bianchi S, Macchiarelli G, Borini A: Ultrastructural markers of quality in human mature oocytes vitrified using cryoleaf and cryoloop. Reprod Biomed Online 2009, 19(Suppl 3):17-27.
• [24]Camboni A, Martinez-Madrid B, Dolmans MM, Amorim CA, Nottola SA, Donnez J, Van Langendonckt A: Preservation of fertility in young cancer patients: contribution of transmission electron microscopy. Reprod Biomed Online 2008, 17:136-150.
• [25]Coticchio G, Borini A, Distratis V, Maione M, Scaravelli G, Bianchi V, Macchiarelli G, Nottola SA: Qualitative and morphometric analysis of the ultrastructure of human oocytes cryopreserved by two alternative slow cooling protocols. J Assist Reprod Genet 2010, 27:131-140.
• [26]Khalili MA, Maione M, Palmerini MG, Bianchi S, Macchiarelli G, Nottola SA: Ultrastructure of human mature oocytes after vitrification. Eur J Histochem 2012, 56:e38.
• [27]Shahedi A, Hosseini A, Khalili MA, Norouzian M, Salehi M, Piriaei A, Nottola SA: The effect of vitrification on ultrastructure of human in vitro matured germinal vesicle oocytes. Eur J Obstet Gynecol Reprod Biol 2013, 167:69-75.
• [28]Borini A, Sciajno R, Bianchi V, Sereni E, Flamigni C, Coticchio G: Clinical outcome of oocyte cryopreservation after slow cooling with a protocol utilizing a high sucrose concentration. Hum Reprod 2006, 21:512-517.
• [29]Lassalle B, Testart J, Renard JP: Human embryo features that influence the success of cryopreservation with the use of 1,2 propanediol. Fertil Steril 1985, 44:645-651.
• [30]Rienzi L, Balaban B, Ebner T, Mandelbaum J: The oocyte. Hum Reprod 2012, 27(Suppl 1):i2-i21.
• [31]Borini A, Bonu MA, Coticchio G, Bianchi V, Cattoli M, Flamigni C: Pregnancies and births after oocyte cryopreservation. Fertil Steril 2004, 82:601-605.
• [32]Levi Setti PE, Albani E, Novara PV, Cesana A, Morreale G: Cryopreservation of supernumerary oocytes in IVF/ICSI cycles. Hum Reprod 2006, 21:370-375.
• [33]Bianchi V, Coticchio G, Distratis V, Di Giusto N, Flamigni C, Borini A: Differential sucrose concentration during dehydration (0.2 mol/l) and rehydration (0.3 mol/l) increases the implantation rate of frozen human oocytes. Reprod Biomed Online 2007, 14:64-71.
• [34]Rall WF, Fahy GM: Ice-free cryopreservation of mouse embryos at -196 degrees C by vitrification. Nature 1985, 313:573-575.
• [35]Rienzi L, Cobo A, Paffoni A, Scarduelli C, Capalbo A, Vajta G, Remohí J, Ragni G, Ubaldi FM: Consistent and predictable delivery rates after oocyte vitrification: an observational longitudinal cohort multicentric study. Hum Reprod 2012, 27:1606-1612.
• [36]Vajta G: Vitrification in human and domestic animal embryology: work in progress. Reprod Fertil Dev 2013, 25:719-727.
• [37]Potdar N, Gelbaya TA, Nardo LG: Oocyte vitrification in the 21st century and post-warming fertility outcomes: a systematic review and meta-analysis. Reprod Biomed Online 2014, 29:159-176.
• [38]Kuwayama M, Vajta G, Kato O, Leibo SP: Highly efficient vitrification method for cryopreservation of human oocytes. Reprod Biomed Online 2005, 11:300-308.
• [39]Vajta G, Nagy ZP: Are programmable freezers still needed in the embryo laboratory? Review on vitrification. Reprod Biomed Online 2006, 12:779-796.
• [40]Roy TK, Brandi S, Tappe NM, Bradley CK, Vom E, Henderson C, Lewis C, Battista K, Hobbs B, Hobbs S, Syer J, Lanyon SR, Dopheide SM, Peura TT, McArthur SJ, Bowman MC, Stojanov T: Embryo vitrification using a novel semi-automated closed system yields in vitro outcomes equivalent to the manual Cryotop method. Hum Reprod 2014, 29:2431-2438.
• [41]Stoop D, De Munck N, Jansen E, Platteau P, Van den Abbeel E, Verheyen G, Devroey P: Clinical validation of a closed vitrification system in an oocyte-donation programme. Reprod Biomed Online 2012, 24:180-185.
• [42]Palmerini MG, Antinori M, Maione M, Cerusico F, Versaci C, Nottola SA, Macchiarelli G, Khalili MS, Antinori S: Ultrastructure of immature and mature human oocytes after cryotop vitrification. J Reprod Dev 2014, 60:411-420.
• [43]Boldt J: Current results with slow freezing and vitrification of the human oocyte. Reprod Biomed Online 2011, 23:314-322.
• [44]Gualtieri R, Mollo V, Barbato V, Fiorentino I, Iaccarino M, Talevi R: Ultrastructure and intracellular calcium response during activation in vitrified and slow-frozen human oocytes. Hum Reprod 2011, 26:2452-2460.
• [45]El Shafie M, Sousa M, Windt M-L, Kruger TF: An Atlas of the Ultrastructure of Human Oocytes. New York, USA: Parthenon Publishing; 2000.
• [46]Makabe S, Van Blerkom J, Nottola SA, Naguro T: Atlas of Human Female Reproductive Function. Ovarian Development to Early Embryogenesis after In Vitro Fertilization. London, UK: Taylor & Francis; 2006.
• [47]Motta PM, Nottola SA, Micara G, Familiari G: Ultrastructure of human unfertilized oocytes and polyspermic embryos in an IVF-ET program. Ann N Y Acad Sci 1988, 541:367-383.
• [48]Motta PM, Nottola SA, Makabe S, Heyn R: Mitochondrial morphology in human fetal and adult female germ cells. Hum Reprod 2000, 15(Suppl 2):129-147.
• [49]Motta PM, Nottola SA, Familiari G, Makabe S, Stallone T, Macchiarelli G: Morphodynamics of the follicular-luteal complex during early ovarian development and reproductive life. Int Rev Cytol 2003, 223:177-288.
• [50]Bianchi S, Macchiarelli G, Micara G, Aragona C, Maione M, Nottola SA: Ultrastructural and morphometric evaluation of aged cumulus-oocyte-complexes [abstract]. It J Anat Embryol 2013, 118(2(Suppl)):28.
• [51]Gualtieri R, Iaccarino M, Mollo V, Prisco M, Iaccarino S, Talevi R: Slow cooling of human oocytes: ultrastructural injuries and apoptotic status. Fertil Steril 2009, 91:1023-1034.
• [52]Ebner T, Moser M, Sommergruber M, Gaiswinkler U, Shebl O, Jesacher K, Tews G: Occurrence and developmental consequences of vacuoles throughout preimplantation development. Fertil Steril 2005, 83:1635-1640.
• [53]Familiari G, Heyn R, Relucenti M, Nottola SA, Sathananthan AH: Ultrastructural dynamics of human reproduction, from ovulation to fertilization and early embryo development. Int Rev Cytol 2006, 249:53-141.
• [54]Sathananthan AH, Selvaraj K, Girijashankar ML, Ganesh V, Selvaraj P, Trounson AO: From oogonia to mature oocytes: inactivation of the maternal centrosome in humans. Microsc Res Tech 2006, 69:396-407.
• [55]Sathananthan AH: Ultrastructure of human gametes, fertilization and embryos in assisted reproduction: a personal survey. Micron 2013, 44:1-20.
• [56]Gardner DK, Sheehan CB, Rienzi L, Katz-Jaffe M, Larman MG: Analysis of oocyte physiology to improve cryopreservation procedures. Theriogenology 2007, 67:64-72.