In Vtro Maturation Outcomes of hCG-primed Unstimulated Cycles in Patients with Polycystic Ovarian Syndrome: a Single-center Experience


 Backgound: IVM has emerged as a safe and promising alternative procedure to conventional in vitro fertilization (IVF) for minimizing the risk of ovarian hyperstimulation syndrome (OHSS) in patients with PCOS. Despite the comparable obstetric and perinatal outcomes, there are no definite factors known to affect the outcomes of IVM.Methods: Retrospective analysis of a total of 313 women with PCOS undergoing 427 hCG-primed IVM cycles between January 2010 and February 2016 at the Fertility Center of CHA Gangnam Medical Center, Seoul, Korea. The number of retrieved oocytes and maturation, fertilization, and implantation rates were analyzed. Results: After transferring a mean of 2.4 ± 0.5 fresh embryos, the clinical pregnancy rate was 39.1% (n = 167), and the live birth rate was 30.7% (n = 131) with the implantation rate of 20.9%. The numbers of retrieved (18.1 ± 9.7 vs. 15.6 ± 8.7, p = 0.014), fertilized (8.6 ± 5.2 vs. 6.6 ± 3.8, p < 0.001) oocytes; good-quality embryos (1.3 ± 0.9 vs. 1.0 ± 0.9, p = 0.001); and blastocyst transfer cycles (22 vs. 15, p < 0.001) were significantly higher in the live birth group than in the no live birth group. Among the factors associated with live births, retrieved oocytes had a slightly positive effect on live birth (RR = 1.03; 95% CI, 1.00, 1.06; p = 0.021).Conclusions: It seems that the number of retrieved oocytes has a favorable effect in increasing the clinical pregnancy rate and live birth rate during hCG-primed IVM procedure in women with PCOS. Physicians’ skills and cautious efforts may be required to retrieve a higher number of oocytes in IVM procedures.


Background
Subfertility occurs in 15-20% of couples trying to conceive, and ovulatory factor account for up to 30-40% of all cases of female subfertility. Polycystic ovarian syndrome (PCOS) affects 4-7% of all women of reproductive age and 50% of those presenting with subfertility [1]. A certain proportion of women with PCOS require assisted reproductive technology (ART). Occasionally, controlled ovarian hyperstimulation in women with PCOS is associated with risks of complications such as ovarian hyperstimulation syndrome (OHSS), resulting in cycle cancellation or freeze-all strategies.
In vitro maturation (IVM) has emerged as a safe alternative procedure to conventional in vitro fertilization (IVF) for minimizing the risk of OHSS in patients with PCOS. Cha et al. reported the rst successful pregnancy resulting from IVM of unstimulated ovaries from donors [2], and Trounson et al. initially achieved an IVM baby from the mother's autologous oocytes [3]. Since then, IVM has also been successfully used in a wide range of subfertile populations, especially in women with PCOS. So far, more than 5,000-6,000 IVM babies have been born worldwide [4]. The American Society for Reproductive Medicine (ASRM) Committee announced that the IVM procedure should be performed only as an experimental procedure evaluating both e cacy and safety in carefully selected patients in 2013 [5].
However, a recent meta-analysis found that the clinical pregnancy rate was higher with the IVM procedure than with conventional IVF although with very low-quality evidence, investigating randomized controlled trials in patients with PCOS [1]. More recently, Yu et al. reported no signi cant increased risk in obstetric, neonatal, and childhood development associated with IVM after a mean follow-up of 7.5 years compared with conventional IVF in women with PCOS in a retrospective case-control study [6].
Several researchers have developed and used different kinds of protocols for IVM procedures: IVM with FSH priming, which is a short course usage of gonadotropins to increase oocyte yield, or IVM with hCG priming to facilitate the resumption of meiosis in vivo before full oocyte maturation [7]. According to some studies, hCG priming increased the maturation, fertilization, and pregnancy rates from 69% to 84%, 45% to 80%, and 31% to 51.9%, respectively, while FSH priming increased the pregnancy rate by up to 29% [8, 9].
Although IVM is considered a promising alternative procedure in women with PCOS, most studies have focused on the e cacy and safety, especially compared with conventional IVF. Several studies have tried to investigate the potential factors to improve IVM procedures, such as the size of follicles before oocyte retrieval, use of either FSH or hCG priming, optimal timing for hCG injection in hCG-primed cycles, and components in culture media [10,11]. However, currently, there is no de nite factor that has been proven to improve IVM outcomes.
The aim of this study was to investigate the factors that are related to successful IVM outcomes in women with PCOS during hCG-primed unstimulated cycles.

Study population
This retrospective study investigated the parameters from medical charts of 313 patients with PCOS who underwent IVM cycles between January 2010 and February 2016 at the Fertility Center of CHA Gangnam Medical Center, Seoul, Korea. A total of 427 IVM cycles were included in this study. PCOS was diagnosed based on the consensus meeting of the European Society of Human Reproduction and Embryology (ESHRE) and ASRM [12]; if patients had other etiologies such as congenital adrenal hyperplasia, androgen-secreting tumors, Cushing's syndrome, or neoplastic disease, they were excluded. This study was approved by the Institutional Review Board of CHA Gangnam Medical Center (GCI 2020-03-006). IVM protocol, oocyte retrieval, and culture procedure All patients underwent unstimulated cycles with hCG priming. Oocyte retrieval was performed between menstrual cycle days 9 and 33, based on the patient's cycle length after serial checking of antral follicle count (AFC) and endometrial thickness by transvaginal ultrasonography. The patients were subcutaneously injected with recombinant hCG (choriogonadotropin-α, 250 µg, Ovidrel, Merck Serono, Germany) 35-36 h before oocyte retrieval. The immature oocytes were retrieved with a transvaginal ultrasound guide using a 17-gauge double-lumen aspiration needle (Cook, Australia) with a suction pressure of 160 mmHg.
IVM procedures were performed at 37°C, 5% CO2, and 5% O2 in the incubator. After the oocytes were cultured for 24-48 h, they were denuded with hyaluronidase solution (ICSI Cumulase; Origio). The maturity of the oocytes was microscopically determined by the presence of the rst polar body. Mature oocytes were inseminated by ICSI to initiate fertilization and embryonic development. At 66-120 h after ICSI, one to three embryos were selected according to the number and quality of embryos and transferred into the patient's uterus. Supernumerary embryos were cultured and frozen when they reached the blastocyst stage. A good-quality embryo at the cleavage stage was de ned as a 4-cell embryo on day 2 and a 7-or 8-cell embryo on day 3, contained <20% anucleate fragments, and exhibited no apparent morphological abnormalities. As for blastocyst-stage embryos, a good-quality embryo was de ned according to the Gardner and Schoolcraft criteria [13].
For endometrial preparation, patients were given oral estradiol valerate (Progynova, Schering Pharmaceuticals) with a daily oral dose of 4 mg, starting from the day of oocyte retrieval. Luteal support was also provided by both progesterone injection (daily, 50 mg, Watson Pharmaceuticals) and transvaginal progesterone (daily, 600 mg of Utrogestan, Piette International Laboratories; 300 mg of Lutinus, Ferring Pharmaceuticals; or 300 mg of Endometrin, Ferring Pharmaceuticals) starting from the day of fertilization until 8 weeks of gestation, if a patient was pregnant. Serum β-hCG concentrations were measured 11-12 days after embryo transfer (ET). Clinical pregnancy refers to the identification of a pregnancy sac in the uterus at 6 weeks of gestation. After the con rmation of clinical pregnancy, women were followed up for pregnancy outcomes.

Measurement of demographic and clinical outcomes
All data were collected using electronic medical records, including baseline characteristics (age, body mass index (BMI), AFC, and duration of infertility) and basal hormone levels (anti-Müllerian hormone (AMH), FSH, LH, and estradiol). Parameters related to IVM cycles were also investigated, such as the number of retrieved oocytes and maturation, fertilization, implantation, and clinical pregnancy rates.

Statistics
Data were analyzed using SPSS 26.0 (IBM, USA). Statistical analysis was performed using the chi-square test, Student t-test, and multivariate regression analyses. Differences were considered signi cant when the P value was less than 0.05.

Results
Baseline patient characteristics are summarized in TABLE 1. The mean age at undergoing IVM was 33.1 ± 3.4 years, having the mean duration of infertility of 3.7 ± 2.4 years. All of 313 women in this study had PCOS with the mean AMH of 10.9 ± 5.6 ng/mL, having the mean AFC of 30.4 ± 15.0 on hCG day.

Discussion
This study found that the numbers of retrieved, total matured, and fertilized oocytes and day of ET after oocyte retrieval were signi cantly higher in favor of live births during hCG-primed IVM cycles in women with PCOS.
The overall clinical pregnancy (39.1%) and live birth (30.7%) rates in this study were comparable to previous reports of 20.4% and 68.5% of the clinical pregnancy rate and 18.8%-33.7% of the live birth rate [8, 14,1,4]. The ET with blastocyst group showed a signi cantly higher clinical pregnancy rate (67.6%) than the ET without blastocyst group after transferring a mean number of 2.0 blastocysts. In a previous study of hCG-primed IVM in women with PCOS in 2007, the clinical pregnancy rate of blastocyst ET was also higher than that of cleavage ET (51.9% vs. 28.4%), following the transfer of a mean number of 2.95 blastocysts [15]. Therefore, the results of the present study imply more evolved and promising aspects of IVM procedures via recent developments in ART departments. These studies indicated that AFC was a signi cant predictor of the number of oocytes retrieved. The present study did not show a signi cant difference in the AFC on hCG day between the live birth and no live birth groups. However, the number of retrieved oocytes and AFC on hCG day were signi cantly higher in the ET with blastocyst group than in the ET without blastocyst group.
Considering the ndings of the present study, it may be important to try to retrieve more oocytes for higher pregnancy rates as well as higher blastocyst formation. For this, it is worrisome about patients' pain, intraperitoneal bleeding, and other possible complications. Seyhan et al. recently reported that oocyte retrieval was well tolerated and did not seem to be associated with more complications than IVF although IVM required more punctures per ovary [19]. The number of retrieved oocytes was much smaller than that of AFC on hCG day in the present study. Therefore, physicians' skills and cautious efforts are needed to retrieve a higher number of oocytes in IVM as well as in IVF.
To increase the clinical pregnancy rate, some authors in previous studies have insisted that IVM should be scheduled when the follicles are larger than 12 mm in diameter for better oocyte competence [10], whereas other researchers have suggested retrieving the leading follicles sized <12 or <14 mm in IVM cycles [20,9,21,22]. However, the diameter of the leading follicles on hCG did not differ between the live birth and no live birth groups in this study, with a mean diameter under 10 mm in both groups.
Endometrial thickness is also an important parameter in ART, but there are only a few studies that have analyzed the effect of endometrial thickness in IVM procedures. The present study showed no signi cant difference between the live birth and no live birth groups in terms of endometrial thickness (overall mean thickness, 7.0 mm). It is known that endometrial development may be suboptimal in IVM compared with that in natural or stimulated cycles [8]. In this regard, Ortega-Hrepich et al. suggested successful pregnancy after warming vitri ed embryos in FSH-primed IVM cycles without hCG priming, adopting hormone replacement ET cycle [23]. The present study used hCG priming and endometrial preparation with su cient hormone therapy. Several researchers have suggested that hCG priming is bene cial in terms of endometrial synchronization with the developing embryo as well as oocyte yield and maturational competence [24]. The patients in this study were prescribed hormonal medications for endometrial preparation, including oral estradiol with vaginal and intramural progesterone, whereas most studies used oral estradiol and vaginal progesterone [24]. Therefore, it is possible to suggest that hCG priming combined with su cient luteal supplement is bene cial for relatively thinner endometrium in women in fresh IVM cycles.
This study has a few limitations. This was a retrospective study with a relatively small number of patients. However, to our knowledge, this is the largest study analyzing the factors associated with hCGprimed unstimulated IVM outcomes. Further prospective studies with larger numbers of patients are needed to con rm the results of this study.

Conclusion
It seems that the number of retrieved oocytes has a favorable effect in increasing the clinical pregnancy rate and live birth rate during hCG-primed IVM procedure in women with PCOS. In conclusion, it seems that the number of retrieved oocytes has a favorable effect on the live birth rate during hCG-primed IVM procedures in women with PCOS.

Declarations
Ethics approval and consent to participate This study was approved by the Institutional Review Board of CHA Gangnam Medical Center (GCI 2020-03-006).

Consent for publication
Not applicable.

Availability of data and materials
The datasets during and/or analysed during the current study available from the corresponding author on reasonable request.