A total of 65 FF samples from 54 women were collected. The inclusion criteria were as follows: 1) age<42 yr; 2) normal ovulatory cycle with cycle lengths between 25 and 35 days; 3) body mass index<32 kg/m²; and 4) no evidence of endocrine abnormalities such as hyperprolactinemia, thyroid dysfunction, or polycystic ovarian syndrome as defined by the Rotterdam criteria (17). Patients with stage III-IV endometriosis were excluded. FF was obtained from the largest follicle sized≥20 mm of mean diameter from each ovary. FF containing no oocyte or contaminated with blood was excluded.

All women received ovarian stimulation using gonadotropin releasing hormone (GnRH) antagonist multiple-dose flexible protocols. Recombinant FSH (Gonal-F; Serono, Geneva, Switzerland) was started on the third menstrual cycle day without oral contraceptive pretreatment. The GnRH antagonist, cetrorelix (0.25 mg Cetrotide; Serono), was added daily, starting when the diameter of the leading follicle reached 14 mm and ending when the mean diameter of the leading follicle reached 18 mm or when 2 or more follicles had a diameter of 17 mm. Recombinant human chorionic gonadotropin (hCG) (250 µg Ovidrel; Serono) was administered subcutaneously 36 hr before transvaginal oocyte retrieval. Up to 3 embryos were transferred 3 days after oocyte retrieval. The luteal phase was supported with 50 mg of progesterone in oil (Progest; Samil, Seoul, Korea) or 8% progesterone gel (Crinone; Serono) daily, initially for 14 days starting on the day of oocyte retrieval and continuing for another 6 to 8 weeks in cases where a pregnancy was achieved. Pregnancy was first assessed using serum β-hCG levels 14 days after oocyte retrieval, and clinical pregnancy was defined by the presence of an intrauterine gestational sac with pulsating fetal heart beats.

FF was obtained from the largest follicle sized≥20 mm of mean diameter from each ovary during oocyte retrieval. We punctured the largest follicle and aspirated the fluid into an empty bottle without any media. After the needle was withdrawn, it was flushed to obtain the possible trapped oocyte in the dead space of the collection needle. Samples were collected from only 1 follicle for each ovary, and only 1 needle was used for each follicle. Collected samples were centrifuged at 500×g for 15 min; FF supernatant was stored at -20℃ until assayed.

FF AMH levels were measured using enzyme-linked immunosorbent assay (Immunotech Beckman Coulter, Marseille, France). The measurement range was 0.14-21 ng/mL. Intra- and inter-assay coefficients of variation were 12.3% and 14.2%, respectively, with a sensitivity of 0.14 ng/mL.

Samples were divided into 3 groups according to the FF AMH levels: below the 33th percentile (low group, FF AMH<2.1 ng/mL, n=21), between the 33th and the 67th percentile (intermediate group, FF AMH=2.1-3.6 ng/mL, n=22), or above the 67th percentile (high group, FF AMH>3.6 ng/mL, n=22).

Oocytes were placed in the insemination medium (Sydney IVF fertilization medium; Cook Women's Health, Spencer, IN, USA) 1 to 4 hr before insemination with 50,000 to 500,000 motile spermatozoa per milliliter of medium. Intracytoplasmic sperm injection was performed in our institution according to the following criteria: 1) total motile sperm count<50,000, 2) retrieval of fewer than 3 oocytes, or 3) a history of total failed fertilization. Fertilization was assessed 16 to 18 hr after insemination by the presence of 2 pronuclei and a second polar body. The fertilized oocytes were maintained in the culture medium (Sydney IVF cleavage medium; Cook Women's Health).

Normal fertilization was defined as the presence of 2 pronuclei and a second polar body. Top-quality embryos were designated as grade A or B embryos with at least 6 blastomeres on day 3 after fertilization. The embryo grades were classified according to the our laboratory criteria: A - no blastomeres fragmentation; B - fragmentation less than 20%; C - fragmentation between 20% -50% anucleate fragments, irregularity of blastomeres, and no apparent morphologic abnormalities; D - fragmentation above 50%. Embryo score was calculated as the points of embryo grade multiplied by the number of blastomeres. Four points were given for embryo grade A, 3 for grade B, 2 for grade C, and 1 for grade D.

Data were analyzed with SPSS software version 21.0 (SPSS, Chicago, IL, USA). Data were compared by Student's t-test, analysis of variance (ANOVA), or chi-square test, as indicated. Correlation between the FF AMH level and day 3 embryo score was determined by bivariate correlation analysis with Pearson's correlation coefficients. The result was considered as significant when the P value was <0.05.

This study protocol was reviewed and approved by the institutional review board of Seoul National University Bundang Hospital (IRB No. B-0801/053-016). Informed consent was obtained from all patients.

Clinical characteristics of the study subjects and COS outcomes are presented in Table 1. Comparing the characteristics and COS outcomes among the low, intermediate, and high groups, no significant differences were observed except for basal FSH and numbers of oocytes retrieved.

FF AMH levels correlated positively with the matched embryo score on day 3 after fertilization (r=0.331; P=0.015) (Fig. 1).

The normal fertilization rate was significantly lower in the low group than in the intermediate group (61.9% vs. 95.5% vs. 77.3%, in the low, intermediate, and high groups, respectively, P=0.028). The top-quality embryos rate showed the slight increases in the groups but differences did not reach statistical significance (23.8% vs. 45.5% vs. 50.0%, respectively, P=0.175) (Fig. 2).