P1: GXB Journal of Assisted Reproduction and Genetics PP960-jarg-471269 August 14, 2003 14:53 Style file version June 3rd, 2002 Journal of Assisted Reproduction and Genetics, Vol. 20, No. 9, September 2003 (C 2003) Assisted Reproduction The Effect of Preincubation Period of Oocytes on Nuclear Maturity, Fertilization Rate, Embryo Quality, and Pregnancy Outcome in IVF and ICSI1 Jason Yen-Ping Ho,2 Ming-Jer Chen,2,3 Yu-Chiao Yi,2 Hwa-Fen Guu,2 and Esther Shih-Chu Ho2 Submitted December 5, 2002; accepted May 28, 2003 Purpose: To clarify the effect of preincubation of oocytes on the results of IVF and ICSI. Methods: A total of 176 IVF and 64 ICSI cycles received long protocol ovarian stimulation. The oocytes were incubated for 1–8 h before insemination or sperm injection. Metaphase II (MII) percentage was evaluated in the ICSI arm; fertilization rates, embryo quality, and pregnancy outcomes were analyzed in both IVF and ICSI arms according to the preincubation period duration of oocytes. Results: The MII percentage of the ICSI arm was significantly lower (P < 0.05) in the group with preincubation period of <2.5 h. The fertilization rates in groups with preincubation for 2.5–5.5 h were significantly higher (P < 0.001) for IVF. Embryo quality and pregnancy outcomes were not significantly different between the IVF or ICSI arm. Conclusions: The preincubation of oocytes for at least 2.5 h is beneficial to both IVF and ICSI outcomes by increasing the nuclear maturity of oocytes. KEY WORDS: Fertilization rates; ICSI; IVF; metaphase II; preincubation period. INTRODUCTION bation in IVF arose because some studies showed no effect of preincubation of oocytes on fertilization rate Delayed insemination in human in vitro fertilization or pregnancy outcome unless the preincubation pe- (IVF) was originally introduced by Trounson et al. (1) riod was longer than 9 h (5,6). in 1982 in a study demonstrating better fertilization When intracytoplasmic sperm injection (ICSI) was rates of oocytes and higher embryo quality when the first introduced in 1992, many procedures were based retrieved oocytes received preinsemination incuba- on previous experience with IVF, including the con- tion. This method was later adopted by most IVF cen- cept of preincubation of oocytes (7). In the begin- ters producing the best IVF results when oocytes were ning, preincubation for 3–7 h was adopted in most inseminated 4–6 h after retrieval (2–4). The preincu- centers (8,9), but later studies contradicted this opin- bation of oocytes was considered to complete the fi- ion. Although improved fertilization rate after ICSI nal nuclear and cytoplasmic maturation of the oocyte. on preincubated human oocytes was previously de- However, controversy about the necessity of preincu- scribed (10), other researchers supported opposing conclusions (11–13). Complete nuclear and cytoplasmic maturation of 1 Presented in part at the PRSFS (Pacific Rim Society for Fertility and Sterility), 2002. oocytes is essential for the activation of oocytes at 2 Department of Obstetrics and Gynecology, Taichung Veterans fertilization and the development of embryos (14). In General Hospital, 160, Sec. 3, Chung-Kang Road, Taichung 407, a natural cycle, the lutenizing hormone (LH) surge Taiwan, Republic of China. 3 To whom correspondence should be addressed; e-mail: k388@ initiates the resumption of meiosis in the oocyte and vghtc.vghtc.gov.tw. hence completes the final maturation of the oocyte. In 1058-0468/03/0900-0358/0 C 2003 Plenum Publishing Corporation 358 P1: GXB Journal of Assisted Reproduction and Genetics PP960-jarg-471269 August 14, 2003 14:53 Style file version June 3rd, 2002 Effect of Preincubation on Oocytes 359 the stimulating cycle, human chorionic gonadotropin in a cycle with IVF failure; and (iv) blastocyst trans- (hCG) has been used to induce oocyte maturation in fer; (v) inadequate documentation of preincubation a controlled environment. An oocyte is considered to period; (vi) different stimulation protocol. The pa- reach nuclear maturity when its meiosis is arrested tient selection criteria for ICSI were (i) zero or very again at metaphase II (MII) with the presence of an low normal fertilization rate (less than 20%) in two extruded first polar body. Because cytoplasmic matu- previous standard IVFs; (ii) poor sperm parameters rity may not be synchronous with nuclear maturity in (lower than 5 105 progressively motile spermato- the stimulating cycle (15,16), the fertilizing ability of zoa with normal morphology) after sperm prepara- an oocyte with a mature nucleus is not necessarily at tion; and (iii) testicular sperm. A total of 176 IVF maximum potential. and 64 ICSI cycles from 180 patients were included in In IVF cycles, the level of nuclear maturity is usually this analysis. The patients were categorized into five unknown because the oocytes are surrounded by cu- groups in each of the IVF and ICSI arms according to mulus and corona cells. On the other hand, denuding the period between oocyte retrieval and insemination procedures must be performed before ICSI to remove or injection: Group I, <2.5 h; Group II, 2.5 to <3.5 h; surrounding cumulus and corona cells and to ascertain Group III, 3.5 to <4.5 h; Group IV, 4.5 to <5.5 h; nuclear maturity. As a result, factors other than nu- Group V, 5.5 h. clear maturity seem to affect the fertilizing ability of oocytes in ICSI since only MII oocytes receive sperm Sperm Preparation injection. During the 3-year course of this study, oocyte re- Semen samples were collected at the time of trievals were performed uniformly 34 h after hCG oocyte retrieval by masturbation after 2–5 days of administration. The insemination or injection proce- abstinence. The semen parameters were evaluated dures were performed randomly 1–8 h after oocyte according to published protocols (17), including retrieval. Because all patients enrolled in this study (i) manual evaluation of sperm concentration and received the same ovarian stimulating protocol, we percentage motility by counting with a Makler cham- assumed that the same preincubation period in the ber according to World Health Organization (1992) IVF arm would result in a similar degree of nuclear criteria; (ii) evaluation of sperm vitality using the maturity to that which would occur in the ICSI arm conventional eosin–nigrosin stained smear; and (iii) if the dosage of gonadotropin and ovarian response evaluation of sperm morphology using Kruger’s strict were similar. To elucidate how preincubation would criteria (Diff–Quik stain). The sample was layered influence nuclear maturity, we retrospectively ana- onto a discontinuous Pure-Sperm (Nidacon Interna- lyzed the MII oocyte percentage within the ICSI arm tional, Gothenburg, Sweden) two-layer gradient (40 according to varying oocyte preincubation periods. To and 80%) and centrifuged at 300g for 30 min. The further clarify the effect of the duration of the preincu- pellet was washed twice with IVFTM-20 (IVF Science bation period on the prognosis, we conducted a ret- Scandinavia, Gothenburg, Sweden) for insemination. rospective analysis on the fertilization rate, embryo quality, and pregnancy outcome in IVF and ICSI cy- IVF and ICSI Protocols cles with varying oocyte preincubation periods. In all cycles, ovarian stimulation was carried out by a downregulation protocol using gonadotropin- MATERIALS AND METHODS releasing hormone analogue (GnRHa) leuprolide ac- etate (Lupron , Takeda Chemical Industries, Os- Subjects aka, Japan) followed by gonadotropins including hu- We retrospectively analyzed data collected from man menopausal gonadotropin (hMG; Pergonal , patients who received IVF or ICSI between January FSH 75 IU and LH 75 IU in each ampoule, Serono, 1999 and December 2001 at the Department of Ob- Aubonne, Switzerland) and follicle-stimulating hor- stetrics and Gynecology, Taichung Veterans General mone (FSH; Metrodin , FSH 75 IU, Serono, Hospital, Taiwan. Patients registered for IVF or ICSI Aubonne, Switzerland). The patient received 0.5 mg were enrolled in this study, except for those with the of GnRHa by daily subcutaneous injection starting following conditions: (i) embryo transfer using pre- at the mid-luteal phase of the previous cycle. Reduc- vious cryopreserved embryo; (ii) failure to retrieve tion of GnRHa dosage to 0.25 mg daily and 4–6 am- oocytes; (iii) fertilization using rescue ICSI technique poules of gonadotropin daily was started on Day 3 Journal of Assisted Reproduction and Genetics, Vol. 20, No. 9, September 2003 P1: GXB Journal of Assisted Reproduction and Genetics PP960-jarg-471269 August 14, 2003 14:53 Style file version June 3rd, 2002 360 Ho, Chen, Yi, Guu, and Ho of the menstrual cycle. Gonadotropin dosage was ad- Pregnancy test using serum hCG assay was per- justed accordingly by monitoring the follicles using formed on Day 12 after embryo transfer. A serial rise transvaginal ultrasound starting on Day 9 of the men- in serum hCG concentration would suggest a posi- strual cycle. Ovulation was triggered by using 10,000 tive pregnancy outcome. Ongoing pregnancy refers to IU hCG (Profaci , Serono, Aubonne, Switzerland) successful progress of pregnancy beyond the twelfth when two leading follicles reached a mean diameter week of gestation. of 18 mm. Oocyte retrieval was performed 34 h after hCG administration. Statistics The cumulus–corona–oocyte complexes were pooled and incubated in IVF medium. In the case of Statistical analysis was performed with SPSS sta- ICSI, the oocytes received denudation procedures us- tistical software (SPSS v 5.0 for Windows). Statistical ing enzymatic digestion with 80 IU/mL hyaluronidase significance was assessed using the Student’s t test, 2 and capillary pipette dissection just before the in- test, and one-way analysis of variance (ANOVA). At jection of sperm. Insemination of oocytes in IVF P < 0.05, the difference was considered to be statisti- was performed with spermatozoa adjusted to a con- cally significant.