Relationship between follicular size and developmental capacity of oocytes under controlled ovarian hyperstimulation in assisted reproductive technologies

Abstract Purpose We investigate the relationships between oocyte developmental capacity and follicular size of its origin in Japanese women: those undergoing conventional IVF (cIVF) and ICSI, respectively. Methods A total of 3377 follicles were punctured separately and were classified into three groups (large, medium, and small) by their diameters. A total of 1482 retrieved oocytes were individually cultured and received cIVF or ICSI. The oocytes receiving ICSI were denuded and the number of mature (MII) oocytes was counted. Results The oocyte retrieval rates and the proportion of MII oocytes were significantly lower in small follicles than in large follicles. Under cIVF, the fertilization rate was significantly lower in oocytes from small follicles than large follicles. Under ICSI, the fertilization rate for MII oocytes was not significantly related to follicular size. Follicular size was not significantly related to the development potential to blastocyst and pregnancy rate for either the cIVF oocytes or the ICSI oocytes. Conclusions Although the fertilization rate by cIVF is low in oocytes from small follicles due to the lower proportion of mature oocytes, their development potential is comparable to that of oocytes from larger follicles if they could be fertilized. Under ICSI using mature oocytes, their development potential is not related to follicular size.

under FSH stimulation. 4 Therefore, there is a possibility that the developmental capacity of oocytes retrieved during COH depends on the size of the follicles from which they came. In fact, previous reports showed that the oocyte retrieval rate was lower for small follicles than it was for large follicles. 5,6 Some reports have shown that oocytes from small follicles have a reduced rate of fertilization or embryo development. [6][7][8][9] However, other studies did not find such differences. 5,10,11 Therefore, the relationship between follicular size and developmental capacity of oocytes is still controversial.
These discordant findings might be due to differences in patient characteristics, methods of oocyte insemination (conventional IVF [cIVF] or intracytoplasmic sperm injection [ICSI]), and the assessment of developmental capacity. In most previous studies, the developmental capacity of oocytes has been evaluated by assessing the cleavage rate or the quality of cleavage-stage embryos, but not blastocysts. However, blastocyst transfer is becoming more common because it results in a higher implantation rate than does the transfer of cleavage-stage embryos. 12 Because blastocyst quality is strongly associated with the pregnancy rate, 13  Furthermore, because the efficiency of ART is different between races, 15,16 it remains unclear whether these findings also apply to Japanese patients. According to the latest report from the International Committee Monitoring Assisted Reproductive Technologies (ICMART), Japan is the largest user of ART worldwide in terms of annual number of treatment cycles performed. 1,17,18 Therefore, it is important to conduct a study to clarify the relationship between follicular size and the development capacity of oocytes in Japanese women. In this study, we individually cultured the retrieved oocytes and investigated the size/development relationship by examining blastocyst development and pregnancy rate of oocytes from groups of Japanese women undergoing cIVF or ICSI.

| Study population
This retrospective cohort study included 176 patients with infertility who underwent oocytes retrieval for cIVF or ICSI at Yamaguchi University Hospital between June 2014 and June 2020. Informed consent was obtained from all the patients in this study. The study design was reviewed and approved by the institutional review board of Yamaguchi University Hospital.

| Controlled ovarian hyperstimulation protocol and oocyte retrieval
Controlled ovarian hyperstimulation was performed using standard gonadotropin releasing hormone agonist (GnRHa) / FSH protocols.
Nasal spray GnRHa (900 μg/d) was given from the mid-luteal phase in the previous cycle to continuously suppress pituitary gonadotropin secretion until the injection of HCG (10 000 IU). COH was initiated from the 2nd day of the IVF-ET cycle by injection of 225 IU FSH for 3 days, followed by a daily injection of 150 IU HMG. When more than three leading follicles reached 18 mm or more, HCG was injected for ovulation induction. Oocyte retrieval was carried out 35 hours after HCG injection. For patients who showed poor response to COH in the previous IVF cycle, they underwent short GnRH agonist protocol. In this protocol, COH and GnRHa were simultaneously initiated from the 2nd day of the IVF-ET cycle. 300 IU FSH or 300 IU HMG was daily injected until the day of HCG injection.
Each follicular size was measured and recorded before the oocyte aspiration. Follicles were classified into three groups according to their diameters as measured by transvaginal ultrasonography: large follicle (≧18 mm), medium follicle (13-17 mm), and small follicle (≦12). Each follicle was aspirated separately, and the retrieved oocyte was individually cultured to correlate their developmental outcomes with follicular size. For the oocytes receiving ICSI, oocytes were denudated, and the number of metaphase II (MII) oocytes was counted. Only MII oocytes were proceeded to sperm injection.

| Fertilization and embryo transfer
The semen samples were collected by masturbation, and the motile sperm was collected by swim-up technique as reported previously. 19 Fertilization was performed using either standard insemination (cIVF) or ICSI with Piezo-assisted ICSI system (Prime Tech Ltd). In the early years of this study, all oocytes were subjected to cIVF.
From 2017 onwards, ICSI was introduced to patients with severe male factor or fertilization failure. If the sperm concentration is low (motile sperm concentration is <5 × 10 5 /mL after swim-up) or three was a history of fertilization failure (previous fertilization rate under cIVF was <25%), all retrieved oocytes underwent ICSI procedure.
If the previous fertilization rate under cIVF was between 26% and 50%, half of the retrieved oocytes were allocated to receive ICSI, and the other half were allocated to receive cIVF. This was designated as "split" as reported previously. 20 Fertilization was confirmed on day 1 (17-19 hours after insemination) with the presence of two pronuclei. The cleavage of embryo was assessed on day 2. The blastocyst formation and its quality were assessed on day 5 according to the established score guidelines. 21 A high-quality blastocyst was defined as having a grade of at least 3BB, including 3/4/5AA, AB, BA, or BB. 22 Fresh embryo transfer was performed on either day 2 (cleavage-stage embryo) or day 5 (blastocyst stage). One or two embryos were transferred in each cycle. Blastocysts remaining after embryo transfer were stored at a low temperature using the vitrification technique and used for frozen embryo transfer (FET) in the future cycle. The protocols for FET included the natural cycle and the hormone replacement therapy cycle with transdermal estradiol and vaginal progesterone. The urine hCG test was carried out in 9 days after embryo transfer.

| Outcomes
The numbers of punctured follicles, retrieved oocytes, fertilized oocytes, cleavage embryo, and blastocysts and its quality were evaluated in each of three groups (small, medium, and large follicles).
Oocyte retrieval rate was expressed as a ratio of the number of re-

| RE SULTS
A total of 176 patients (314 cycles) underwent cIVF, ICSI or split, respectively ( Table 1). The prevalence of asthenozoospermia and oligospermia was significantly higher in the ICSI group than in the cIVF group. Consequently, the sperm concentration and sperm motility were significantly lower in the ICSI group than in the cIVF group. There were no significant differences of other backgrounds between three groups. In total, 3377 follicles were individually punctured, and 1482 oocytes were retrieved ( Table 2). As shown in Table 2, the oocyte retrieval rates from small and medium follicles were significantly lower than the retrieval rate from large follicles.
Furthermore, the retrieval rate was lower for small follicles than for medium follicles. Table 3 shows the outcomes of 870 oocytes that underwent cIVF. The fertilization rates were significantly lower for oocytes from small and medium follicles than for oocytes from large follicles. Follicular size did not have a significant effect on the cleavage rate, the formation rate of blastocyst or blastocyst quality. To calculate the pregnancy rate, we analyzed the transfer of one or two embryos derived from the same follicular size group. In total, 144 cycles (162 embryos) were included in the analysis. The pregnancy rate and spontaneous abortion rate were not significantly affected by follicular size. used for sperm injection in ICSI cycle, the fertilization rate was calculated as the ratio of fertilized oocytes to the number of MII oocytes.
The fertilization rate was not significantly affected by follicular size.
As was observed in cIVF-oocytes, follicular size did not have a significant effect on the cleavage rate, the formation rate of blastocyst, blastocyst quality, pregnancy rate, or spontaneous abortion rate.

| D ISCUSS I ON
In this study, by culturing oocytes individually and monitoring the developmental outcomes up to the blastocyst stage, we found that the developmental capacity of fertilized oocytes under both cIVF and ICSI is not related to follicular size in Japanese women. Although small follicles contain fewer mature oocytes than large follicles, they can grow into blastocysts as well as oocyte from large follicles provided that they are fertilized. Therefore, our study shows the usefulness of puncturing not only large follicles, but also small follicles under COH cycle.
We found a significantly lower retrieval rate for oocytes from small follicles than for oocytes from large follicles, which is consistent with previous reports. 5 follicles were more easily detached from the follicular wall by aspiration than were those in smaller follicles.
Under cIVF, the fertilization rare in oocytes from small follicles was significantly lower than that from large follicles, which is consistent with previous reports. 11,24 However, under ICSI, the fertilization rate was not affected by follicular size as reported previously. 11,14 Because oocytes undergoing cIVF were not denuded, their maturity was unclear. On the other hand, oocytes undergoing ICSI needed to be denuded for sperm injection, which made it possible to observe their maturity. In the ICSI cycle, the proportion of mature oocytes was lower for oocytes from small follicles than for oocytes from large follicles. There is no doubt that these proportions were not markedly different in the retrieved oocytes under cIVF cycle. Therefore, the decreased fertilization rate of oocytes from small follicle under cIVF may be due to a lower proportion of mature oocytes. This is supported by previous findings that the proportion of mature oocytes was correlated with follicular size. 5,10,14 In other words, oocytes from small follicles have same potential of fertilization as oocytes from large follicles if they are matured.
Our study also examined whether the developmental capacity of the fertilized oocytes is correlated with the follicular size. Some studies have found a correlation between oocyte developmental capacity and follicular size, 7,9 while others did not. 5,10,11,24 Therefore, their relationship is still controversial. However, these reports assessed it according to the cleavage rate or the quality of cleavage-stage embryos, but not blastocysts. Recently, transfer of human embryo at the blastocyst stage is becoming more common in the practice of assisted reproduction technology because blastocyst it is associated with a higher implantation rate than transfer of cleavage-stage embryos. 12 In addition, it should be noted that only 30% of cleavage-stage embryos can reach the blastocyst stage. 25  This study revealed the relationship between the developmental capacity of oocytes up to blastocyst stage and follicular size under both cIVF and ICSI in Japanese women. Although the fertilization rate by cIVF is low in oocytes from small follicles due to a lower proportion of mature oocytes, their development potential is comparable to that of oocytes from larger follicles if they could be fertilized by either cIVF or ICSI. Therefore, it is worth to puncture small follicles which are grown under COH. Because Japan has the largest number of ART cases in the world, out study should provide useful information to many clinicians and patients.

D I SCLOS U R E
Conflict of interest: All authors have no conflict of interest.
Ethics Statement: Informed consent was obtained from all the patients in this study. The study design was reviewed and approved by the institutional review board of Yamaguchi University Hospital.