McGill Reproductive Center, Department of Obstetrics and Gynecology, McGill University, Montreal, Canada
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: Cryopreservation/immature oocytes/ICSI/pregnancy/PCOS
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
Case report |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
After 24 and 48 h of incubation, mature oocytes were denuded of cumulus cells using finely drawn glass pipettes following 1 min exposure to 0.1% hyaluronidase solution (Medi-Cult, Hopkinton, MA, USA). Spermatozoa for ICSI were prepared by gradient separation (45 and 90% gradients) at 560 g for 20 min. Following gradient separation, the sperm pellet was washed twice (200 g) with 5 ml of IVF medium (Medi-Cult Inc.). After ICSI, each oocyte was transferred to 20 µl of Medi-Cult medium under mineral oil (Sigma) for culture. Fertilization was assessed 1618 h after ICSI for the appearance of two distinct pronuclei (2PN) and two polar bodies. Fertilized 2PN oocytes were transferred to 1.0 ml of Medi-Cult medium in two-well organ tissue culture dishes for a further 24 h. For the preparation of the endometrium, the patient was given 4 mg of oestradiol (Estrace; Roberts Pharmaceutical, Mississauga, Canada) in divided doses starting on the day of oocyte retrieval. Luteal support was provided in the form of 200 mg progesterone (Prometrium) twice a day for 16 days starting 48 h after oocyte collection.
A total of 63 oocytes were retrieved (10 with abnormal morphology, 22 at metaphase-I stage and 31 at germinal vesicle stage). Following maturation in culture for 24 h, 22 oocytes were noted to be at metaphase-II stage. After ICSI of the 22 oocytes, 15 were observed to have 2PN on the second day. Of 15 fertilized oocytes 13 cleaved. A total of 3 embryos (one 3-cell stage grade 1, two 4-cell stage grade 3) were transferred at 48 h following ICSI. At embryo transfer, the endometrial thickness was 8.9 mm at transvaginal ultrasound scan. Embryo transfer was performed using a Wallace catheter (SIMS Protex Limited, Hythe, Kent, UK), and two weeks later the serum ß-HCG level was 122.2 IU/l. Six weeks after embryo transfer an ongoing intrauterine singleton pregnancy with fetal heartbeat was confirmed by transvaginal ultrasonography. Unfortunately, this pregnancy miscarried at eight weeks after embryo transfer.
Following maturation in culture for 48 h of the remaining 31 oocytes, 19 oocytes were at metaphase-II stage. After ICSI of those oocytes, 16 oocytes were observed to have 2PN. These 16 fertilized oocytes were cryopreserved immediately after fertilization assessment. Embryo freezing was performed by the slow freezing protocol (Lassalle et al., 1985). Briefly, 2PN oocytes were suspended for 20 min in 2 ml of cryoprotectant (1.5 mol/l propanediol, 0.1 mol/l sucrose) in Dulbecco's phosphate-buffered saline (PBS; GIBCO BRL, Rockville, MD, USA) containing 30% human serum albumin solution (5% human albumin; Bayer, Elkhart, IN, USA). They were then aspirated into 0.25 ml straws (CryoBioSystem, Orsay, France) in a small central droplet of cryoprotectant surrounded by two air bubbles and adjacent cryoprotectant droplets. The straws were then frozen horizontally in a programmable freezer (Planer Kryo 10 Series III; Planer Products Ltd., Sunbury-on-Thames, UK) using a slow-freezing protocol (2°C/min to 7°C; hold at 7°C for 10 min and manual seeding; 0.3°C/min to 30°C; 50°C/min to 180°C, hold at 180°C for 20 min and then plunge into liquid nitrogen). Thawing was performed by maintaining the straws first in air for 1015 sec and then plunging them into a water bath at 30°C for 30 sec. The central droplet, containing the 2PN oocytes was then gently expelled from the straw in 2 ml of PBS containing 1 mol/l propanediol (PROH) and 0.2 mol/l sucrose. The 2PN oocytes were then washed free of the cryprotectants by sequential incubation (10 min) in three aliquots of PBS solution (2 ml) containing lowered molar concentrations of PROH/sucrose (0.5/0.2; 0/0.2; 0/0).
Standard protocol was followed for the preparation of the endometrium for frozen embryo transfer. Briefly, the patient received intravaginal progesterone (Prometrium) in a dose of 300 mg a night for 10 days to induce a withdrawal bleed. After stopping the progesterone, menstrual bleeding started in 3 days. On day 2 of the cycle, the patient was given 200 µg of the gonadotrophin-releasing hormone agonist, Buserelin (Suprefact Synthetic LH-RH analogue; Hoechst Marion Roussel Canada Inc., Laval, Quebec, Canada) and 6 mg oestradiol (Estrace) daily for 10 days. Two days before embryo transfer, Busrelin was stopped and progesterone was administered vaginally (200 mg twice a day).
Three months after cryopreservation, nine fertilized oocytes were thawed for scheduled embryo transfer. Following thawing, 8 fertilized oocytes survived (88.9%; 8/9) and 7 cleaved (87.5%). Three embryos were transferred (one 4-cell stage grade 2, two 4-cell stage grade 3). The endometrial thickness measured 9.0 mm on the day of embryo transfer. However, after two weeks no pregnancy ensued as confirmed by a negative pregnancy test. The second frozen embryo transfer was performed 5 months later. The remaining 7 fertilized oocytes were thawed and all survived (100%; 7/7). After 24 h of culture, all embryos had cleaved (100%; 7/7). Three embryos were transferred (two 4-cell stage grade 2 and one 4-cell stage grade 3). The endometrial thickness measured 9.5 mm on the day of embryo transfer. Two weeks later, the serum ß-HCG concentration was 258.5 IU/l and 6 weeks after embryo transfer an ongoing intrauterine singleton pregnancy with fetal heartbeat was confirmed by transvaginal ultrasonography. An uneventful delivery at 38.5 weeks resulted in the birth of a normal healthy boy (3040 g).
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
The first report of pregnancy in a woman with anovulatory infertility following IVM of immature oocytes and IVF was made in 1994 (Trounson et al., 1994). Recently, it has been demonstrated that recovery of immature oocytes followed by in-vitro maturation of these immature oocytes is a promising treatment for women with PCOS (Chian et al., 1999
, 2000
). To date, approximately 2530% clinical pregnancy rates were established from immature oocytes retrieved from patients with PCOS following IVM in several infertility centres (Cha et al., 2000; personal communication
). Therefore, these data suggest that women with PCOS-related infertility could be treated with immature oocyte retrieval followed by in-vitro maturation and fertilization, as well as cryopreservation.
Embryos survive and implant at higher rates when frozen at the pronuclear stage compared with the cleavage stage (Quinn, 1990; Demoulin et al., 1991
; Veeck et al., 1993
). Recent reports also show that embryo cryopreservation at the pronuclear stage optimizes the chance for a live born infant from a single oocyte retrieval (Damario et al., 2000
; Senn et al., 2000
). Our results show that embryo survival rates were high following freezethawing at the 2PN stage (88.9% and 100.0%). Our previous results also show that survival rates of embryos produced by in-vitro matured oocytes are higher when frozen at the 2PN stage compared with the cleavage stage (unpublished data). In conclusion, the embryos produced from in-vitro matured oocytes retrieved from unstimulated women with PCOS can be safely cryopreserved and healthy infants can be delivered following embryo transfer.
![]() |
Notes |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Almahbobi, G., Anderiesz, C., Hutchinson, P. et al. (1996) Functional integrity of granulose cells from the polycystic ovaries. Clin. Endocrin., 44, 571580.[ISI]
Barnes, F.L., Kausche, A., Tiglias, J. et al. (1996) Production of embryos from in vitro-matured primary human oocytes. Fertil. Steril., 65, 11511156.[ISI][Medline]
Cha, K.Y. and Chian, R.C. (1998) Maturation in vitro of immature human oocytes for clinical use. Hum. Reprod. Update, 4, 103120.
Cha, K.Y., Han, S.Y., Chung, H.M. et al. (2000) 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., 73, 978983.[ISI][Medline]
Chian, R.C., Gülekli, B., Buckett, W.M. and Tan, S.L. (1999) Priming with human chorionic gonadotrophin before retrieval of immature oocytes in women with infertility due to the polycystic ovary syndrome. N. Eng. J. Med., 341, 16241626.
Chian, R.C., Buckett, W.M., Tulandi, T. and Tan, S.L. (2000) Prospective randomized study of human chorionic gonadotrophin priming before immature oocyte retrieval from unstimulated women with polycystic ovarian syndrome. Hum. Reprod., 15, 165170.
Damario, M.A., Hammitt, D.G., Session, D.R. et al. (2000) Embryo crypreservation at the pronuclear stage and efficient embryo use optimizes the chance for a liveborn infant from a single oocyte retrieval. Fertil. Steril., 73, 767773.[ISI][Medline]
Demoulin, A., Jouan, C., Gerday, C. and Dubois, M. (1991) Pregnancy rates after transfer of embryos obtained from different stimulation protocols and frozen at either pronucleate or multicellular stages. Hum. Reprod., 6, 799804.[Abstract]
Erickson, G.F. and Yen, S.S.C. (1984) New data on follicle cells in polycystic ovaries: a proposed mechanism for the genesis of cystic follicles. Semin. Reprod. Endocrinol., 42, 3943.
Goldzieher, J.W. and Green, J.A. (1962) The polycystic ovary. I. Clinical and histological features. J. Clin. Endocrinol. Metab., 22, 325338.[ISI]
Goldzieher, J.W. and Axelrod, L.R. (1963) Clinical and biochemical features of polycystic ovarian disease. Fertil. Steril., 14, 631653.[ISI]
Jakimiuk, A.J., Jakowicki, J.A. and Magoffin, D.A. (1997) Follicular development in polycystic ovary syndrome. Assis. Reprod. Reviews, 7, 5457.
Lassalle, B., Testart, J. and Renard, J.P. (1985) Human embryo features that influence the success of cryopreservation with the use of 1,2-propanediol. Fertil. Steril., 49, 283289.
MacDougall, M.J., Tan, S.L., Balen, A. et al. (1993) A controlled study comparing patients with and without polycystic ovaries undergoing in-vitro fertilization. Hum. Reprod., 8, 233237.[Abstract]
Quinn, P. (1990) Success of oocyte and embryo freezing and its effect on outcome with in-vitro fertilization. Semin. Reprod. Endocrinol., 8, 272280.[ISI]
Senn, A., Vozzi, C., Chanson, A. et al. (2000) Prospective randomized study of two cryopreservation policies avoiding embryo selection: the pronucleate stage leads to a higher cumulative delivery rate than the early cleavage stage. Fertil. Steril., 74, 946952.[ISI][Medline]
Trounson, A.O., Wood, C. and Kausche, A. (1994) In-vitro maturation and the fertilization and developmental competence of oocytes recovered from untreated polycystic ovarian patients. Fertil. Steril., 62, 353362.[ISI][Medline]
Veeck, L.L., Amundson, C.H., Brothman, L.J. et al. (1993) Significantly enhanced pregnancy rates per cycle through cryopreservation and thaw of pronuclear stage oocytes. Fertil. Steril., 59, 12021207.[ISI][Medline]
Yen, S.S.C. (1980) The polycystic ovary syndrome. Clin. Endocrinol. (Oxf.), 12, 177208.[ISI][Medline]
Submitted on January 12, 2001; accepted on May 16, 2001.