Hum. Reprod. Advance Access originally published online on September 4, 2008
Human Reproduction 2008 23(12):2680-2685; doi:10.1093/humrep/den332
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Selection of the optimal day for oocyte retrieval based on the diameter of the dominant follicle in hCG-primed in vitro maturation cycles
McGill Reproductive Center, Department of Obstetrics and Gynecology, Royal Victoria Hospital, McGill University, Montreal, QC H3A 1A1, Canada
1 Correspondence address. Fax: +1-514-843-1496. E-mail: weon-young.son{at}muhc.mcgill.ca
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Abstract |
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BACKGROUND: The efficiency of in vitro maturation (IVM) techniques is suboptimal compared with controlled ovarian stimulation combined with IVF cycles, and studies are needed to identify factors that predispose IVM cycles to success or failure. We compared the outcome of IVM cycles with different dominant follicle (DF) size at oocyte retrieval following hCG priming.
METHODS: IVM was performed in 160 patients with polycystic ovaries (171 cycles). We administered 10 000 IU hCG s.c. 35–38 h before oocyte collection when endometrial thickness reached at least 6 mm. IVM cycles were retrospectively analyzed according to DF diameter as follows; Group 1: DF diameter 
10 mm, Group 2: between 10 and 14 mm, Group 3: >14 mm.
RESULTS: A positive correlation was observed between DF size and number of in vivo matured oocytes collected (Group 1, 2 and 3 = 6.9, 10.6 and 15.1%, respectively). The rates of IVM, fertilization and embryo development were similar among the sibling immature oocytes collected from the three groups. However, clinical pregnancy rate in Group 2 (40.3%) was higher than Group 3 (17.1%) (P < 0.05). Moreover, implantation rates in Groups 1 (13.6%) and 2 (14.3%) were higher than Group 3 (4.9%) (P < 0.01).
CONCLUSIONS: Our results suggest that oocyte collection in IVM cycles should be performed when the DF is 14 mm diameter or less. Sibling immature oocytes may be affected detrimentally if a DF >14 mm is present at oocyte collection.
Key words: dominant follicle diameter/hCG/human immature oocyte/in vitro maturation
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Introduction |
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Many successful pregnancies have recently been reported in cycles of unstimulated in vitro maturation (IVM), which is a simplification of the standard IVF approach utilizing ovarian stimulation (Chian et al., 2000
; Son et al., 2007; Edwards, 2007). However, the efficiency of current IVM techniques is still suboptimal compared with controlled ovarian hyperstimulation (COH) combined with IVF cycles. Therefore, more studies are needed in order to identify factors that predispose these IVM cycles to success or failure.
Compared with standard IVF cycles, there is no universal or consensus protocol for optimal timing of oocyte collection in IVM cycles. Generally, investigators think that the maturation potential of the oocytes and their subsequent fertilization and embryonic development may be affected by endocrine changes that occur in the remaining cohort after selection of a dominant follicle (DF). There has been conflicting evidence regarding the importance of a DF on the day of aspiration before IVM (Russell, 1998; Cobo et al., 1999; Chian et al., 2004). Some investigators have shown a benefit in performing retrieval after the leading follicle reaches 10 mm (Russell, 1998; Mikkelsen et al., 1999), whereas other investigators believed it is detrimental and propose canceling the cycle (Cobo et al., 1999; Le Du et al., 2005). Recently, Chian et al. (2004) suggested that the maturational and developmental competence of sibling immature oocytes was not adversely affected by the presence of any size of DF during the follicular phase.
Therefore, it is still unclear from the published studies the optimum time for collecting oocytes based on DF size in IVM cycles. In addition, there is not enough information concerning the relationship between clinical outcome and DF diameter in hCG-primed IVM cycles. Therefore, this study was performed to compare the embryological differences and clinical outcome in IVM cycles with different sizes of DF at oocyte retrieval following hCG priming.
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Materials and Methods |
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The initial IVM study was approved by the Institutional Review Board of the Hospital. The IVM treatment was explained to the patients, and informed consent was obtained.
Patients
A total of 160 patients (171 cycles) who underwent IVM treatment from June 2005 to March 2008 were included in this retrospective study. Only those patients who had polycystic ovaries (PCO) or polycystic ovary syndrome (PCOS) were recruited. Women with amenorrhea received vaginal 300 mg of progesterone (Prometrium, Schering, Pointe-Claire, QC, Canada) once daily for 10 days to induce withdrawal bleeding. A baseline ultrasound was obtained for all women between Days 2 and 4 of menstrual bleeding to ensure that no ovarian cysts were present and to measure the antral follicle count (AFC) (Tan et al., 2002). Transvaginal ultrasound scans were repeated on Day 8 of the cycle or on the day of hCG administration.
Oocyte collection
Oocyte retrieval was performed between Days 8 and 21 of the menstrual cycle based on the length of the patient's cycle. hCG (10 000 IU) was administered when endometrial thickness reached at least 6 mm (Chian et al., 2000; Son et al., 2008a). The largest follicle and endometrial thickness were measured by ultrasound scan just before egg collection. The size of the largest follicle was measured as a mean of two measurements in a two-dimensional plane. The thickest endometrial segment (between the two interfaces of the endometrial–myometrial junction) was measured transvaginally on a ‘frozen’ midplane, longitudinal section of the uterus by two-dimensional ultrasonography. Oocyte retrieval was performed 35–38 h after hCG priming. Transvaginal ultrasound-guided collection of oocytes was performed using a 19-gauge aspiration needle (K-OPS-7035-RWH-ET, Cook, Australia) with a reduced aspiration pressure of 7.5 kPa. The aspirates were collected in tubes with prewarmed heparinized saline. The oocyte collection was started from the DF and the needle was flushed completely with saline, and oocyte maturity was assessed. Subsequent collection of oocytes without follicle size measurement was performed according to accessibility of the follicles, after which the maturity level of the oocyte was identified. To avoid the possibility of missing oocytes with a small amount of cumulus cells, the remaining follicular aspirates were filtered using 70-µm mesh (Falcon, Becton Dickinson & Company, NJ, USA), washed three times with oocyte wash medium (Cooper Surgical, CT, USA) that contained HEPES buffer supplemented with recombinant human serum albumin, and the oocytes isolated under a stereomicroscope.
In vitro maturation
The nuclear maturity of the collected oocytes was assessed under the dissecting microscope with high magnification (x80) using the sliding method (Son et al., 2006, 2008a). If no germinal vesicle (GV) was observed in the oocyte cytoplasm, the cumulus masses were removed with hyaluronidase and mechanical pipetting after the oocyte collection was completed, and reassessment of oocyte maturity was performed. Oocytes that were matured on the collection day (Day 0: 0–6 h) were inseminated on the same day, while the immature oocytes [GV- or germinal vesicle breakdown (GVBD)-stage] were cultured in IVM medium (Cooper Surgical, CT, USA) supplemented with 75 mIU/ml FSH and LH. Following culture on Day 1 (24–30 h), the oocytes were denuded of cumulus cells with hyaluronidase and mechanical pipetting. After examination, immature oocytes remaining at GV- or GVBD-stage were further cultured in the same medium and the meiotic status was re-examined on Day 2 (48–52-h culture).
IVF, in vitro development and embryo transfer
Matured oocytes were inseminated by ICSI using the partner's spermatozoa. ICSI was performed at least 1 h after observing first polar body (PB) extrusion as suggested by Hyun et al. (2007). Fertilization was assessed 17–19 h after insemination for the appearance of two distinct pronuclei and two PBs. The zygotes were cultured in Embryo Maintenance Medium (Cooper Surgical). Embryonic development was assessed on Day 2 (41–43 h) and on Day 3 (65–67 h) after insemination according to the regularity of blastomeres, the percentage and pattern of anucleate fragments, and all dysmorphic characteristics of the embryos. For this study, we defined embryos as good quality if they had at least a 3-cell embryo on Day 2 and a 6-cell embryo on Day 3, contained <20% anucleate fragments and exhibited no apparent morphological abnormalities. Embryos showing blastomere multi-nucleation, poor cell adhesion, uneven cell division and cytoplasmic abnormalities were defined as low quality. The best quality embryos were transferred on Day 2 or Day 3 after ICSI.
Endometrium preparation
For endometrial preparation, patients received estradiol valerate (Estrace; Roberts Pharmaceutical, Mississauga, Canada), starting on the day of oocyte retrieval. If the endometrial thickness was 6–8 mm, a 8–10 mg dose was given, and if it was 
8 mm, a 6 mg dose was administered, all in divided doses. Luteal support was provided by administering 50 mg of progesterone daily i.m. starting on the day of ICSI and continued, along with estradiol valerate, until 12 weeks of gestation, if the pregnancy test was positive.
Data analysis
IVM cycles were retrospectively analyzed according to the diameter of the DF at the time of collection as follows: Group 1 (n = 63), the DF diameter was 10 mm; Group 2 (n = 67), the DF diameter was between >10 and 14 mm (10< to14 mm): Group 3 (n = 41), the DF diameter was >14 mm. There was similar distribution among the three groups for patient age, duration of infertility, endometrial thickness at retrieval, collection day and number of transferred embryos. However, the mean largest follicle diameter at the time of collection was different (Table I). The number of cycles with PCOS patients was higher in Group 1 (34.9%) than those of Group 2 (17.9%) and Group 3 (9.8%), but there was no difference between Group 2 and Group 3 (P = 0.38). The mean AFC and the number of oocytes collected in Group 1 were significantly higher than those of Group 2 and Group 3 (Table I). Clinical outcome was analyzed in Groups 1–3 according to the origin of transferred embryos as follows; Group A: cycles with only IVM embryos transferred if only immature oocytes, or no oocytes, were collected from DF, or failed fertilization/poor embryo quality of the in vivo matured oocytes collected from DF, and Group B: cycles with transferred embryos derived from oocytes matured in vivo and in vitro.
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In order to clarify the optimal time for hCG priming in IVM cycles, the DF growth occurring between hCG priming and oocyte retrieval was checked in some IVM cycles (n = 94), as the last ultrasound scan to decide the oocyte collection time was not always carried out on hCG priming day, as previously mentioned. The growth of the DF was assessed according to the diameter of
10, 10< to 12 or >12 mm on hCG priming day.
Statistical analysis
Statistical analyses were performed using the 
2, Fisher's exact or t-test as appropriate. All P-values quoted are two-sided, and values <0.05 indicate statistical significance. Analyses were performed using the Statistical Package for the Social Sciences statistical package (SPSS, Inc., Chicago, IL, USA).
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Results |
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Table II summarizes the embryological aspects of in vivo matured oocytes in hCG-primed IVM cycles of Groups 1, 2 and 3. On the day of oocyte retrieval, the rate of in vivo matured oocytes positively correlated with the DF size (Group 1 = 6.9%, Group 2 = 10.6%, Group 3 = 15.1%) (P < 0.01). There was no difference in fertilization, cleavage and good-quality embryos among in vivo matured oocytes collected from the three groups. Table III shows the outcome of oocyte IVM, fertilization, cleavage and embryo development of immature oocytes collected from the three groups. Although a different size of DF was present in each of the three groups, there was no significant difference on IVM and embryonic developmental potential at cleavage stage among the sibling immature oocytes.
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The clinical pregnancies and implantations were analyzed in detail following transfer of the embryos derived from oocytes matured in vivo or in vitro in hCG-primed IVM cycles (Table IV). The clinical pregnancy rate in Group 3 (17.1%) was significantly lower than that of Group 2 (40.3%) (P < 0.05). The clinical pregnancy rate in Group 1 (27.0%) also tended to be higher than that of Group 3 (17.1%), although the difference did not reach statistical significance (P = 0.3). In addition, the implantation rate in Group 3 (4.9%) was significantly lower than that of Group 1 (13.6%) and Group 2 (14.3%) (Table IV) (P < 0.01). In Group 1 and Group 3, higher clinical pregnancy rates were observed when the embryos transferred derived from in vivo matured oocytes rather than from in vitro matured oocytes (36 versus 21.1% in Group 1 and 25 versus 5.9% in Group 3) (Table IV). However, in Group 2, the clinical pregnancy rate was comparable between cycles with and without transferring the embryos produced from in vivo matured oocytes (41.7 versus 38.7%) (Table IV).
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As described in Table IV, 17 pregnancies out of 63 were established in Group 1, which included 4 miscarriages (23.5%, 4/17) and 2 ongoing and 11 term deliveries (17 infants). In Group 2, 27 pregnancies out of 67 were established, which included 7 miscarriages (25.9%, 7/27), 7 ongoing and 13 term deliveries (18 infants). In Group 3, 2 cycles of 7 pregnancies had miscarriages (28.6%, 2/7), 3 ongoing pregnancies and 2 term deliveries with 2 infants.
In Fig. 1, we described the DF growth from hCG priming to the time of oocyte retrieval. Although not statistically significant, the follicle tended to grow differently depending on its size on the day of hCG priming. When the DF size was 10, 10< to 12 or >12 mm at hCG priming, the follicle size was increased by an average 0.5 ± 0.6, 1.4 ± 1.1 and 2.3 ± 1.4 mm, respectively.
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Discussion |
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This study demonstrated that in hCG-primed IVM cycles higher pregnancy and implantation rates can be achieved when the diameter of the DF is 14 and <14 mm at the time of oocyte collection. Our clinical data suggest that the sibling immature oocytes will be affected if a DF over 14 mm is present at the time of oocyte collection, even though no effect is observed on the embryological aspects such as IVM, fertilization and embryo development.
In hCG-primed IVM cycles, a number of new approaches have been recently attempted to improve IVM success rates, such as extending the interval between hCG administration and oocyte collection to 38 h (Son et al., 2008b), or the addition of adjuvant metformin (an insulin sensitizing drug) treatment (Wei et al., 2008) before immature oocyte retrieval in PCOS patients. Nevertheless, the pregnancy and implantation rates after IVM is still lower compared with conventional COH cycles. Therefore, further research is needed to define the best conditions for both clinical and laboratory procedures, such as the timing of immature oocyte collection, culture conditions and the endometrial preparation.
As very little is known about the cellular events involved in recruitment and selection of follicles, no specific marker has yet been developed to determine the optimal time for oocyte collection in unstimulated cycles. However, the decrease of serum FSH concentration at mid-cycle caused by the negative feedback from increasing estradiol, as well as inhibin A concentrations from the leading follicle, plays a major role in ovulation of the selected follicle (Ginther et al., 2001). While the decreased FSH level is still high enough to support the growth of the DF, the rest of the cohort is deprived of FSH and subsequently become atretic. Therefore, the most common view shared by investigators for the optimal timing of immature oocyte collection is based on the presence of a DF. As some of the oocytes in early atretic follicles still possess the competence to support embryonic development (Barnes and Sirard, 2008), it is important to find optimal oocyte retrieval timing between the events causing enhancement of oocyte competence (early atresia) and complete atresia. Although the DF can be distinguished from other cohort of follicles by size of >10 mm diameter (Pache et al., 1990; Fauser and van Heusden, 1997), there has been conflicting evidence regarding the size of a DF on the day of aspiration before IVM. Some investigators reported that the immature oocytes retrieved from a cohort of small follicles could produce viable embryos in the cycles, where the DF size is 14 mm (Russell, 1998; Son et al., 2005) or even 19 mm (Chian et al., 2004). Based on those reports, therefore, we divided the hCG-primed IVM cycles into three groups according to DF diameter at the time of collection to try to establish the optimal time for oocyte retrieval based on the DF diameter.
In the present study, there was no difference in the embryological aspects of in vivo matured oocytes collected from the three groups in terms of fertilization and embryo developmental competence (Table II). The number of oocytes collected in Group 1 was higher than in Group 2 and Group 3, probably due to higher number of antral follicles. In addition, the rates of IVM, fertilization and good embryo development at cleavage stage of sibling immature oocytes did not differ among the three groups (Table III). It appeared that the maturational and developmental potential of sibling immature oocytes were not adversely affected by the presence of a DF during the follicular phase. However, higher clinical pregnancy and implantation rates were achieved in Groups 1 and 2, where oocytes were collected when DF size was 14 mm compared with Group 3, where the DF size was >14 mm (Table IV). We analyzed the clinical outcome according to the origin of the embryos transferred in the three groups and observed a positive correlation between clinical outcome and the presence of transferable embryos derived from the in vivo matured oocytes (Table IV). A better pregnancy rate (35.3%) was obtained in cycles where the transferred embryos also derived from in vivo matured oocytes (Group B) than in cycles without any embryos produced from in vivo matured oocytes (Group A, 24.4%) (Table IV). Interestingly, although the largest follicle diameter was 10 mm in Group 1, a 47.6% (30/63) of the cycles had in vivo matured oocytes (data not shown) and achieved better pregnancy rates (36.0%, 9/25) with embryos produced from the in vivo matured oocytes than that of only IVM embryos (21.1%, 8/38) (Table IV). However, in Group 2, the clinical pregnancy rate was comparable between the cycles with transferred embryos generated from at least one in vivo matured oocyte (41.7%, 15/36) and with only IVM embryos (38.7%, 12/31). In addition, of the three patients that had mixed embryos with one embryo produced from in vivo matured oocytes and IVM embryos transferred, each had a twin pregnancy. These results indicate that sibling immature oocytes in Group 2 generated embryos that have implantation potential and thus, they might have been retrieved just after induction of atresia but before prolonged exposure to the possibly detrimental endocrine and paracrine effects of the DF. Therefore, the clinical pregnancy rate in Group 2 (40.3%) was better than in Group 1 (27.0%), even though implantation rate was similar in the two groups (Table IV).
In contrast, in Group 3, all seven pregnancies were achieved with a singleton pregnancy and six of them had at least one embryo transferred which had been derived from in vivo matured oocytes (25.0%, 6/24). On the other hand, no pregnancy was observed when only IVM embryos were transferred, owing to failure to collect oocytes from DF or failure of in vivo matured oocytes collected from DF to fertilize (0%, 0/14), suggesting that the presence of a DF >14 mm adversely affects the cohort of small follicles. In the other three cycles, only GV-stage oocytes were collected from the DF (15.2, 16 or 16 mm) and a patient was pregnant after transferring embryos produced from only IVM oocytes (data not shown). It is tempting to speculate that the DF that contained a GV-stage oocyte was not able to suppress the rest of the small follicles. These results suggest that the clinical outcome relied most likely on the presence of in vivo matured oocytes, as in a natural cycle, and the sibling immature oocytes are affected adversely when a DF of over 14 mm is present.
Our results are similar to those of Mikkelsen et al. (2000), who observed no differences in IVM, fertilization, cleavage or pregnancy rates in IVM retrievals occurring when the leading follicle was either <12 or 12 mm in women with regular cycles. In addition, our data is in agreement with that of Son et al. (2007), who described 
50% pregnancy rate in patients with PCO where the transferred blastocysts produced from sibling immature oocytes were collected when the largest follicle size was 14 mm in hCG-primed IVM cycles. Paulson et al. (1994) also reported that all pregnancies occurred in the cycles in which one of the embryos was derived from the DF (17%, 11/66) in unstimulated IVF cycles of women who had regular cycles and all implantations were singleton. No pregnancy was achieved in the cycles where only IVM embryos were transferred (0%, 0/10). In the Paulson et al. (1994) study, the mean DF diameter on day of hCG was about 19 mm.
Some investigators (Thornton et al., 1998; Chian et al., 2004; Lim et al., 2007) suggested that sibling immature oocytes exposed to any size of a DF in unstimulated cycles could contribute to the overall pregnancy success. In the Thornton et al., (1998) report, they obtained two pregnancies (a fresh cycle and a frozen cycle) from human IVM oocytes retrieved after mid-cycle aspiration of oocytes following hCG priming in unstimulated cycles with a DF, although the diameter of the DF at the time of oocyte retrieval was not mentioned in the study. In a series case report of Chian et al. (2004), only immature oocytes from an unstimulated IVF cycle were retrieved in one case because no oocyte was collected from the DF (19 mm), and a successful pregnancy was achieved. The other two pregnancies involved the mixed transfer of embryos that resulted from a combination of in vivo matured and IVM oocytes. In the Lim et al. (2007) studies, they observed reasonable pregnancy and implantation rates (29.3 versus 10.4%) after performing unstimulated IVF cycles in patients (n = 123) who had a regular cycle. In their study, most pregnancies were achieved from the cycles that had in vivo matured oocytes (36.3%, 33/91). When patients had only in vitro matured oocytes, the pregnancy rate was only 9.4% (3/32). In that study, the DF diameter on day of hCG varied from <12 to >17 mm. Those reports indicated that in unstimulated IVF cycles the immature oocytes retrieved could contribute to the overall pregnancy success even after being exposed to a DF.
Based on the present study, our data strongly suggest that the best time to collect the oocytes is when a DF reaches between 10 and 14 mm. Therefore, it was important to know when was the best time for hCG priming. For this purpose, we evaluated the follicle growth from hCG priming to oocyte retrieval. The DF size was increased by average 0.5, 1.4 and 2.3 mm when the diameter was 10, 10<to12 and >12 mm on hCG priming day, respectively (Fig. 1). These observations indicate that the hCG should be given to patients when the largest follicle reaches 10–12 mm. For those anovulatory PCOS patients, where the follicles will not grow to 10 mm, oocyte collection can take place when largest follicle size is 10 mm since pregnancy and implantation rates observed in Group 1 are reasonable (27.0 and 13.6%, respectively).
In conclusion, our results demonstrate that the size of the DF at the time of oocyte collection is correlated to the clinical outcome in hCG-primed IVM cycles. Our data strongly suggest that once selection of the leading follicle has occurred, the implantation potential of embryos derived from sibling immature oocytes is impaired, even though there was no difference observed in fertilization and embryo development. Follicular growth must be monitored carefully to ensure that oocyte retrieval takes place before the follicular diameter exceeds 14 mm in hCG-primed IVM cycles. Since in vivo matured oocytes in hCG-primed IVM cycles can produce better viable embryos than that of in vitro matured oocytes, the hCG should be given to the patient when largest follicle reaches 10–12 mm to ensure the presence of in vivo matured oocytes on the day of collection and to avoid detrimental effects on the sibling immature oocytes. Our results emphasize the concept that the size of the DF at the time of oocyte aspiration plays an important role in the success of pregnancy in hCG-primed IVM cycles.
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Submitted on May 18, 2008; resubmitted on July 25, 2008; accepted on August 1, 2008.
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