Jack Y. J. Huang, M.D., Togas Tulandi, M.D., M.H.C.M., Hananel Holzer, M.D., Seang Lin Tan, M.D., M.B.A., and Ri-Cheng Chian, Ph.D.
Department of Obstetrics and Gynecology, McGill University Health Center, McGill University, Montreal, Quebec, Canada

Objective: To report an additional strategy of fertility preservation, which combines ovarian tissue cryobanking with retrieval of immature oocytes from excised ovarian tissue, followed by in vitro maturation (IVM) and vitri- fication.
Design: Retrospective analysis of case series.

Setting: University teaching hospital.
Patient(s):Women who underwent oophorectomy or ovarian wedge resection before receiving chemotherapy and/or radiotherapy.
Intervention(s): Immature oocyte retrieval, IVM, oocyte vitrification, ovarian tissue cryobanking.

MainOutcomeMeasure(s):Oocytes retrieved from the excised ovarian tissue, oocyte maturation rate, and number of oocytes cryopreserved by vitrification.
Result(s):Four consecutive patients underwent retrieval of immature oocytes from the antral follicles of the ex- cised ovarian tissue. The mean number of immature oocytes recovered was three (1, 3, 4, and 3, respectively). The mean maturation rate following IVM was 79% (100%, 100%, 50%, and 67%, respectively). In total, eight mature oocytes were vitrified.
Conclusion(s):Oocytes can be retrieved from excised ovarian tissue, matured in vitro, and cryopreserved by vitrification. This fertility preservation technique could be combined with ovarian tissue cryobanking. (Fertil SterilÒ 2008;89:567–72. Ó2008 by American Society for Reproductive Medicine.) 

Key Words:Fertility preservation, cryopreservation, oocyte, in-vitro maturation, vitrification, ovarian tissue cryobanking

Advances in cancer treatment have improved the long-term survival rates of young women suffering from malignancies (1). However, cancer treatment carries adverse side effects, including loss of ovarian function and sterility. For young women affected by malignancy and autoimmune disorders, fertility preservation options are limited (2).

According to the 2005 Ethics Committee of the American Society for Reproductive Medicine, the only established method of fertility preservation is embryo cryopreservation (3). However, most young patients do not have this option be- cause they either lack a male partner or do not have sufficient time to undergo ovarian stimulation, oocyte retrieval, and in vitro fertilization (IVF) before starting cancer treatment. In addition, some women may not wish to cryopreserve em- bryos for personal, religious, or moral reasons.

Received December 11, 2006; revised and accepted March 27, 2007. Presented at the Annual Meeting of the American Society for Reproductive Medicine, October 21–25, 2006; New Orleans, Louisiana. Abstract P-485.
Reprint requests: Ri-Cheng Chian, Ph.D., Women’s Pavilion F3, Royal Victoria Hospital, 687 Pine Avenue West, Montreal, Quebec, Canada H3A 1A1 (FAX: 514-843-1662; E-mail: ri-cheng.chian@muhc.mcgill.ca).

Ovarian tissue cryobanking has been the primary method of fertility preservation for prepubertal girls who are at risk of premature ovarian failure (4). Ovarian tissue cryopreservation for future transplantation may be offered as a method of fertility preservation for pubertal patients who cannot receive ovarian stimulation due to time constraints or contraindications. However, this procedure remains experimental (5) because there have been only three reported live births from transplantation of cryopreserved ovarian tis- sues (6–8). Furthermore, cryobanking of ovarian tissue only preserves the primordial and primary follicles, as the pre- ovulatory antral follicles, which contain immature oocytes at the germinal vesicle (GV) stage, do not survive the procedure (9).

Oocyte cryopreservation by vitrification is a promising new technique and appears to be more effective than conven- tional slow freezing method (10, 11). In clinical studies using the conventional slow-freezing/rapid thawing method, the survival rates of frozen oocytes averaged 50% and varied markedly between the reported cases and series (23% to 89%) (12). However, over the past 5 years, several groups have reported high survival rates (89.2% to 100%) and successful live births using vitrification (13–18). In fact, since the first report in 1999, vitrification of oocytes has resulted in more than 30 live births worldwide (12).

An attractive strategy of fertility preservation for young women undergoing ovarian cryobanking is retrieval and in vitro maturation (IVM) of immature GV-stage oocytes followed by oocyte vitrification. Ovarian tissues can be excised by laparoscopy, and aspiration of the visible antral follicles from the tissue can be performed. The objective of our study is to report an additional method of fertility preservation that combines ovarian tissue cryobanking with retrieval of immature oocytes from excised ovarian tissue followed by IVM and vitrification of oocytes.

MATERIALS AND METHODS

The four patients were part of a cohort of 22 patients who, since 1997, have undergone ovarian tissue cryopreservation for fertility preservation at the McGill Reproductive Center (MRC) in Montreal, Canada. The indications for ovarian cryobanking included Hodgkin lymphoma (n 1⁄4 2), breast cancer (n 1⁄4 1), and rectal cancer (n 1⁄4 1). The Hospital Research Ethics Board approved the study, and written consent was obtained from all patients.

A laparoscopic ovarian wedge resection of approximately 2.5 X 2 X 1 cm was performed in each of three patients. One patient underwent laparoscopic oophorectomy. Laparoscopic ovarian transposition using a previously described technique (19) was performed in the patient with rectal carcinoma, who was subsequently treated with pelvic irradiation. A single surgeon performed all of the laparoscopic surgeries. In all cases, an ovarian tissue sample was submitted for histopathologic examination. To date, none of the four patients has requested transplantation of the cryopreserved ovarian tissue.

Retrieval of Immature Oocytes from Excised Ovarian Tissue
The excised ovarian tissue was suspended in cold (4°C) Leibovitz L-15 medium (GIBCO/Invitrogen, Carlsbad, CA) and transferred to the laboratory (Fig. 1A). Before dissecting the ovarian cortical tissue for cryopreservation, we aspirated all visible follicles with an 18-gauge syringe needle that was at- tached to a 10-mL syringe (Fig. 1B). The aspirates were

flushed in Oocyte Washing Medium (CooperSurgical/ SAGE, Trumbull, CT) and searched for cumulus–oocyte complexes under the dissecting microscope. Following folli- cle aspiration, the ovarian cortical tissues were isolated in the Oocyte Washing Medium, cut into 4 mm3 (2 X 2 X 1) pieces, and searched for the presence of additional oocytes. The total time required to perform follicle aspiration and immature oocyte retrieval was approximately 15 minutes.

In Vitro Maturation of Oocytes

The retrieved immature oocytes were incubated in an Organ Tissue Culture Dish (60 X 40 mm; Falcon/BD Biosciences, San Jose, CA) containing 1.0 mL of IVM-Medium (Cooper- Surgical) supplemented with a final concentration of 75 mIU/ mL of follicle-stimulating hormone (FSH) and luteinizing hormone (LH) at 37ºC in an atmosphere of 5% CO2 in air with high humidity (20, 21). After maturation in culture for 24 hours, the cumulus cells surrounding the oocytes were re- moved using finely drawn glass pipettes after 1 minute of exposure to 0.1% hyaluronidase solution (Cook, Brisbane, Australia). The mature, metaphase 2 stage (MII) oocytes were cryopreserved by vitrification. The remaining immature oocytes were cultured for an additional 24 hours; if any oocytes became mature, they were similarly vitrified.

a) The excised ovarian tissue was suspended in cold (4ºC) Leibovitz L-15 medium. (b) The visible follicles were aspirated with an 18-gauge syringe needle that was attached to a 10-mL syringe.

FIGURE 1

Huang. Cryopreservation of ovarian tissue and oocytes. Fertil Steril 2008.

Vitrification of Mature Oocytes

Oocyte vitrification was performed because the four patients did not have a male partner and refused insemination of oo- cytes using donor sperm. The in vitro matured oocytes (IVM oocytes) were cryopreserved using a Vitrification Kit purchased from Medicult A/S (Jyllinge, Denmark), and the vitrification method was as previously described elsewhere (22). For the vitrification procedure, the oocytes were suspended in equilibration medium (EM) containing 7.5% (v/v) ethylene glycol (EG) þ 7.5% (v/v) 1,2-propanediol (PROH) for 5 minutes at room temperature, and subsequently transferred to the vitrification medium (VM) containing 15% (v/v) EG þ 15% (v/v) PROH þ 0.5 M sucrose) at room temperature for 45 to 60 seconds. They were then loaded on the McGill Cryoleaf and plunged immediately into liquid nitrogen (LN2) for storage.

Cryopreservation of Ovarian Tissue

Cryopreservation of the ovarian cortical tissue was performed as previously described elsewhere (23). Following follicle aspiration, the ovarian cortical tissue was transferred to a Petri dish containing 5 mL of cold (4ºC) Lebovitz L-15 medium. The ovarian cortical tissue was isolated, cut into 4 mm3 (2 X 2 X 1) pieces, and transferred into 2-mL cryovials containing 1 mL of 1.5 M dimethyl sulfoxide (DMSO), 0.1 M sucrose, and 10% (v/v) human serum albumin in Leibovitz L-15 medium at 4ºC. The ovarian tissue was equilibrated in the cryoprotectant media for 30 minutes at 4ºC. The cryovials were subsequently frozen using a programable freezer (Planer Kryo 360; Planer PLC, Middlesex, United Kingdom) with a cooling rate of 2ºC/minute from 4ºC to – 9ºC. At -9ºC, seeding was manually performed using forceps that had been pre-cooled in LN2. Cooling resumed at a rate of 0.3ºC/minute from -9ºC to -40ºC and 10ºC/minute from -40ºC to -140ºC, at which time the cryovials were plunged into LN2.

RESULTS
Immature Oocyte Retrieval before Ovarian Tissue Cryobanking
Immature oocyte retrieval from the excised ovarian tissue was performed in four consecutive patients (Table 1). The median number of immature oocytes aspirated from the ovarian follicles was three (three, one, four, and three, respectively). In pa- tient number 2, who was 35 years of age, oophorectomy was performed on day 19 of her menstrual cycle; only one immature was recovered. In patient number 4, who was 38 years old, an ovarian wedge resection was performed on day 14 of her cycle, and three GV-stage oocytes were recovered from the excised ovarian tissue. In younger patients, a greater number of immature oocytes may be retrieved from the ex- cised ovarian tissue. In patients number 1 and 3, who were 21 and 18 years old, respectively, ovarian wedge resections were performed on days 2 and 5 of the menstrual cycle, re-

spectively; three and four GV-stage oocytes (Fig. 2) were re- trieved from the biopsied ovarian tissue, respectively.

FIGURE 2

Four immature cumulus–oocyte complexes were retrieved from the excised ovarian tissue.

Huang. Cryopreservation of ovarian tissue and oocytes. Fertil Steril 2008.

Oocyte Maturation Rate

Following IVM, the mean maturation rate was 79% (100%, 100%, 50%, and 67% respectively).

Number of Oocytes Cryopreserved by Vitrification

From the four patients, a total of eight IVM mature oocytes were vitrified (3, 1, 2, and 2 oocytes, respectively).

DISCUSSION

Immature oocytes can be retrieved successfully from the visible antral follicles of excised ovarian tissue, matured in vitro, and cryopreserved by vitrification, providing an alternative method of fertility preservation. This is the first report of combining ovarian tissue cryobanking with vitrification of IVM oocytes. Furthermore, in a scenario where very few oocytes have been obtained using the conventional transvaginal aspiration method, retrieval of immature oocytes from ovarian tissue before cryopreservation represents an additional source of oocytes for fertility preservation.

At the MRC, the efficacy of oocyte vitrification was con- firmed in a recent pilot study involving 15 women, demonstrating a postthaw survival rate of 94%, an implantation rate of 20.4%, and a pregnancy rate of 46.7% (15). These results have led us to expand our fertility preservation strategy to include this novel combination of IVM oocyte vitrification and ovarian tissue cryobanking. Nonetheless, owing to the experimental nature of this technique, the retrieval of immature oocytes from visible antral follicles before ovarian tissue cryobanking was attempted only in the four most recent patients.

The goal of cryobanking ovarian tissue is to preserve the primordial and primary follicles because they more resistant to freezing and comprise 70% to 90% and 20% to 30% of ovarian follicles, respectively (24, 25). However, secondary and antral follicles, which account for about 7% of the follicular population, do not survive the cryopreservation procedure (9). Possible explanations for their poor tolerance to cryopreservation include their complex cellular structure, leading to incomplete cryoprotectant penetration, and their large water content, resulting in the formation of ice crystals. In addition, follicular atresia may be hastened after thawing due to ischemic damage to the ovarian vasculature (26).

The collection of immature oocytes during ovarian tissue cryopreservation was first reported in a study by Revel et al. in 2003 (27), during which immature oocytes were retrieved from the ovarian tissues of seven patients with differ- ent types of cancer. In three patients, IVM of GV-stage oocytes was performed, resulting in a mean maturation rate of 50% (66.7%, 50.0%, and 33.3%). However, in this series, rather than being cryopreserved, the IVM oocytes were inseminated by intracytoplasmic sperm injection. In two patients, the resultant embryos were cryopreserved. In the four other patients, immature GV-stage oocytes were cryopre- served using the 0.3 M sucrose, slow-freezing protocol (28).

Isachenko et al. (29) reported IVM of immature oocytes retrieved during ovarian tissue dissection in two women suffer- ing from Hodgkin lymphoma and International Federation of Gynecology and Obstetrics (FIGO) stage 2a cervical cancer, respectively. Eighteen immature oocytes were recovered. Following IVM, 10 oocytes reached MII stage (maturation rate of 56.3%). Nine oocytes were cryopreserved using the conventional slow-freezing protocol (30).

For those patients who wished to preserve their fertility by ovarian tissue cryobanking, oophorectomy was preferred over ovarian wedge resection because a larger number of an- tral follicles are present; hence, more GV-stage oocytes may be retrieved. However, most of these patients preferred to retain both of their ovaries, and, because of various psychological and social concerns, they were reluctant to agree to the removal of an entire ovary.

In the case of ovarian wedge resection, the selection of smooth cortical tissues, which are devoid of antral follicles, has been proposed to increase the probability of obtaining a high number of primordial follicles (31). Although a large population of primordial follicles can be preserved by cryobanking ovarian cortical tissue, in vitro culture and the maturation of primordial follicles are still at the early stage of development but remain a promising prospect (32).

The number of immature oocytes retrieved from the antral follicles may vary according to the age of the patient, the size of the excised ovarian tissue, and the day of the menstrual cy- cle. Based on the four patients in the current study, it appears that a greater number of immature oocytes may be retrieved from the excised ovarian tissue of younger patients. There- fore, this new fertility preservation strategy is most beneficial

to young, pubertal women who are at risk for premature ovar- REFERENCES

ian failure. It is interesting that immature oocytes can be retrieved from the visible antral follicles of excised ovarian tissue, regardless of the phase of the menstrual cycle.

In our cohort of four patients, the median number of GV- stage oocytes recovered was three, which is comparable with that reported by Revel et al. (27). It is possible that a greater number of GV-stage oocytes may be recovered from the antral follicles by treating patients with a mild ovarian stimulation protocol before performing ovarian wedge re- section or oophorectomy (33). Further studies are needed to determine whether ultrasonographic measurement of antral follicle count is predictive of the number of oocytes retrieved from excised ovarian tissue.

Immature oocyte retrieval followed by IVM has become an effective option for infertile women with polycystic ovaries or polycystic ovary syndrome, resulting in acceptable clinical pregnancy and implantation rates per embryo transfer (34–38). More than 400 healthy infants have been born to date (39–41). In fact, natural-cycle IVF combined with immature oocyte retrieval followed by IVM has also resulted in reasonable pregnancy and implantation rates among ovulatory women who do not have polycystic ovary syndrome (42). In our small series, a mean oocyte maturation rate of 79% was achieved following IVM, demonstrating that a high percentage of GV-stage oocytes from unstimulated ovaries can attain MII stage using this IVM culture system.

Cryopreservation of immature GV-stage oocytes has been investigated as an alternative to freezing mature MII stage oocytes (43–47). In GV-stage oocytes, the chromatin is diffuse and surrounded by a nuclear membrane. Therefore, cryopreservation of GV-stage oocytes seems to avoid spindle depolymerization and theoretically circumvents the risk of polyploidy and aneuploidies. However, poor maturation and subsequent embryonic development of frozen–thawed GV-stage oocytes were noted to be major problems associated with immature egg freezing (45, 47). In fact, only one human live birth has been reported following cryopreservation of GV-stage oocytes and IVM (48). Therefore, cryopreservation of IVM oocytes remains favored over freezing of immature oocytes (47). Recently, we have demonstrated in animal model studies that IVM oocytes could be successfully cryopreserved by vitrification, resulting in a survival rate of 90%, and that the majority of the thawed IVM oocytes maintained normal meiotic spindle and chromosome alignment and contained euploid number of chromosomes (49). How- ever, further studies are required to investigate the embryonic developmental potential of IVM and vitrified human oocytes.

Immature oocyte retrieval from the antral follicles following ovarian wedge resection or oophorectomy and subsequent IVM and vitrification of those oocytes represent an additional strategy for fertility preservation. In selected patients, this method of fertility preservation can be offered as an adjunct to ovarian tissue cryobanking.

REFERENCES

  1. American Cancer Society. Cancer facts and figures—2006. Atlanta, GA: American Cancer Society:Accessed on March 1, 2007. Available at: http://www.cancer.org/docroot/STT/STT_0.asp; 2006.
  2. Lobo RA. Potential options for preservation of fertility in women. N Engl J Med 2005;353:64–7.
  3. Ethics Committee of the American Society for Reproductive Medicine. Fertility preservation and reproduction in cancer patients. Fertil Steril 2005;83:1622–8.
  4. Moffa F, Biacchiardi CP, Fagioli F, Biasin E, Revelli A, Massobrio M, et al. Ovarian tissue cryostorage and grafting: an option to preserve fertility in pediatric patients with malignancies. Pediatr Hematol Oncol 2007;24:29–44.
  5. Practice Committee of the American Society for Reproductive Medicine; Practice Committee of the Society for Assisted Reproductive Technology. Ovarian tissue and oocyte cryopreservation. Fertil Steril 2006;86(Suppl):S142–7.
  6. Donnez J, Dolmans MM, Demylle D, Jadoul P, Pirard C, Squifflet J, et al. Livebirth after orthotopic transplantation of cryopreserved ovarian tissue. Lancet 2004;364:1405–10.
  7. Meirow D, Levron J, Eldar-Geva T, Hardan I, Fridman E, Zalel Y, et al. Pregnancy after transplantation of cryopreserved ovarian tissue in a patient with ovarian failure after chemotherapy. N Engl J Med 2005;353:318–21.
  8. Oktay K. Spontaneous conceptions and live birth after heterotopic ovarian transplantation: is there a germline stem cell connection? Hum Reprod 2006;21:1345–8.
  9. Gosden RG. Gonadal tissue cryopreservation and transplantation. Re- prod Biomed Online 2002;4(Suppl 1):64–7.
  10. Kuleshova LL, Lopata A. Vitrification can be more favorable than slow cooling. Fertil Steril 2002;78:449–54.
  11. Oktay K, Cil AP, Bang H. Efficiency of oocyte cryopreservation: a meta- analysis. Fertil Steril 2006;86:70–80.
  12. Huang J, Tan SL, Chian RC. Fertility preservation for female. J Reprod Contracep 2006;17:109–28.
  13. Katayama KP, Stehlik J, Kuwayama M, Kato O, Stehlik E. High survival rate of vitrified human oocytes results in clinical pregnancy. Fertil Steril 2003;80:223–4.
  14. Kuwayama M, Vajta G, Leibo S. Highly efficient vitrification method for cryopreservation of human oocytes. Reprod Biomed Online 2005;11:300–8.
  15. Chian RC, Son WY, Huang JY, Cui SJ, Buckett WM, Tan SL. High survival rates and pregnancies of human oocytes following vitrification: preliminary report. Fertil Steril 2005;84:S36.
  16. Kyono K, Fuchinoue K, Yagi A, Nakajo Y, Yamashita A, Kumagai S. Successful pregnancy and delivery after transfer of a single blastocyst derived from a vitrified mature human oocyte. Fertil Steril 2005;84:1017.
  17. Okimura T, Kato K, Zhan Q, Kuwayama M, Zhang J, Kato O. Update on clinical efficiency of the vitrification method for human oocytes in an in vitro fertilization program [abstract]. Fertil Steril 2005;84:S174.
  18. Lucena E, Bernal DP, Lucena C, Rojas A, Moran A, Lucena A. Success- ful ongoing pregnancies after vitrification of oocytes. Fertil Steril 2006;85:108–11.
  19. Tulandi T, Al-Took S. Laparoscopic ovarian suspension before irradiation. Fertil Steril 1998;70:381–3.
  20. Chian RC, Gulekli B, Buckett WM, Tan SL. Priming with human chorionic gonadotropin before retrieval of immature oocytes in women with infertility due to the polycystic ovary syndrome [published correc- tion appears in N Engl J Med 2000;342:224]. N Engl J Med 1999;341:1624, 1626.
  21. Chian RC, Buckett WM, Tulandi T, Tan SL. Prospective randomized study of human chorionic gonadotrophin priming before immature oocyte retrieval from unstimulated women with polycystic ovarian syn- drome. Hum Reprod 2000;15:165–70.
  22. Chian RC, Kuwayama M, Tan L, Tan J, Kato O, Nagai T. High survival rate of bovine oocytes matured in vitro following vitrification. J Reprod Dev 2004;50:685–96.
  23. Gosden RG. Low temperature storage and grafting of human ovarian tis- sue. Mol Cell Endocrin 2000;163:125–9.
  24. Gougeon A. Dynamics of follicular growth in the human: a model from preliminary results. Hum Reprod 1986;1:81–7.
  25. Lass A, Silye R, Abrams DC, Krausz T, Hovatta O, Margara R, et al. Follicular density in ovarian biopsy of infertile women: a new method to assess ovarian reserve. Hum Reprod 1997;12:1028–31.
  26. Revel A, Schenker J. Ovarian tissue banking for cancer patients: is ovarian cortex cryopreservation presently justified? Hum Reprod 2004;19: 14–9.
  27. Revel A, Koler M, Simon A, Lewin A, Laufer N, Safran A. Oocyte collection during cryopreservation of the ovarian cortex. Fertil Steril 2003;79:1237–9.
  28. Fabbri R, Porcu E, Marsella T, Rocchetta G, Venturoli S, Flamigni C. Human oocyte cryopreservation: new perspectives regarding oocyte survival. Hum Reprod 2001;16:411–6.
  29. Isachenko E, Rahimi G, Isachenko V, Narworth F. In-vitro maturation of germinal vesicle oocytes and cryopreservation in metaphase I/II: a possible additional option to preserve fertility during ovarian tissue cryopreservation. Reprod Biomed Online 2004;8:553–7.
  30. Gook DA, Osborn SM, Johnston WI. Cryopreservation of mouse and human oocytes using 1,2-propanediol and the configuration of the mei- otic spindle. Hum Reprod 1993;8:1101–9.
  31. Donnez J, Martinez-Madrid B, Jadoul P, Van Langendonckt A, Demylle D, Dolmans MM. Ovarian tissue cryopreservation and transplantation: a review. Hum Reprod Update 2006;12:519–35.
  32. Abir R, Nitke S, Ben-Haroush A, Fisch B. In vitro maturation of human primordial ovarian follicles: clinical significance, progress in mammals, and methods for growth evaluation. Histol Histopathol 2006;21: 887–98.
  33. Wynn P, Picton HM, Krapez JA, Rutherford AJ, Balen AH, Gosden RG. Pretreatment with follicle stimulating hormone promotes the numbers of human oocytes reaching metaphase II by in-vitro maturation. Hum Reprod 1998;13:3132–8.
  34. Cha KY, Han SY, Chung HM, Choi DH, Lim JM, Lee WS. Pregnancies and deliveries after in vitro maturation culture followed by in vitro fertilization and embryo transfer without stimulation in women with polycystic ovary syndrome. Fertil Steril 2000;73:978–83.
  35. Chian RC, Buckett WM, Tan SL. In-vitro maturation of human oocytes. Reprod Biomed Online 2004;8:148–66.
  36. Le Du A, Kadoch IJ, Bourcigaux N, Doumerc S, Bourrier MC, Chevalier N, et al. In vitro oocyte maturation for the treatment of infertility associated with polycystic ovarian syndrome: the French experience. Hum Reprod 2005;20:420–4.
  37. Papanikolaou EG, Platteau P, Albano C, Nogueira D, Cortvrindt R, Devroey P, et al. Immature oocyte in-vitro maturation: clinical aspects. Reprod Biomed Online 2005;10:587–92.
  38. Soderstrom-Anttila V, Makinen S, Tuuri T, Suikkari AM. Favourable pregnancy results with insemination of in vitro matured oocytes from unstimulated patients. Hum Reprod 2005;20:1534–40.
  39. Jurema MW, Nogueira D. In vitro maturation of human oocytes for assisted reproduction. Fertil Steril 2006;86:1277–91.
  40. Chian RC, Lim JH, Tan SL. State of the art in in-vitro maturation. CurrOpin Obstet Gynecol 2004;16:211–9.
  41. Mikkelsen A. Strategies in human in-vitro maturation and their clinical outcome. Reprod Biomed Online 2005;10:593–9.
  42. Chian RC, Buckett WM, Abdul Jalil AK, Son WY, Sylvestre C, Rao D, et al. Natural cycle in-vitro fertilization combined with in-vitro maturation of immature oocytes is an alternative approach in infertility treatment. Fertil Steril 2004;82:1675–8.
  43. Mandelbaum J, Junca AM, Plachot M, Alnot MO, Salat-Baroux J, Alvarez S, et al. Cryopreservation of human embryos and oocytes. Hum Reprod 1988;3:117–9.
  44. Mandelbaum J, Belaısch-Allart J, Junca AM, Antoine JM, Plachot M, Alvarez S, et al. Cryopreservation in human assisted reproduction is now routine for embryos but remains a research procedure for oocytes. Hum Reprod 1998;13(Suppl 3):161–77.
  45. Toth TL, Baka SG, Veeck LL, Jones HW Jr, Muasher S, Lanzendorf SE. Fertilization and in vitro development of cryopreserved human prophase I oocytes. Fertil Steril 1994;61:891–4.
  46. Toth TL, Lanzendorf SE, Sandow BA, Veeck LL, Hassen WA, Hansen K, et al. Cryopreservation of human prophase I oocytes collected from un- stimulated follicles. Fertil Steril 1994;61:1077–82.
  47. Son WY, Park SE, Lee KA, Lee WS, Ko JJ, Yoon TK, et al. Effects of 1,2- propanediol and freezing on the in vitro developmental capacity of hu- man immature oocytes. Fertil Steril 1996;66:996–9.
  48. Tucker MJ, Wright G, Morton PC, Massey JB. Birth after cryopreservation of immature oocytes with subsequent in vitro maturation. Fertil Steril 1998;70:578–9.
  49. Huang JY, Chen HY, Wang Y, Tan SL, Chian RC. Meiotic spindle and chromosome alignment of in-vitro matured oocytes following vitrification [abstract]. Fertil Steril 2006;86(Suppl 1):S66.