The development of controlled ovarian hyperstimulation (COH), which allows multiple oocytes to grow and mature simultaneously, has paved the way for in vitro fertilization (IVF) –embryo transfer.1 After decades of clinical practice and investigation, several COH protocols have been developed for IVF, and can be classified into two types, conventional pituitary downregulation protocol and others.2–5 Women who receive IVF treatment can be classified as having poor, normal, or high ovarian responsiveness to gonadotropin (Gn). Patients with poor ovarian responses (PORs) are least likely to get pregnant via IVF treatment.6 One of the main causes resulting in the poor response is diminished ovarian reserve (DOR).7,8 DOR is characterized by increased follicle-stimulating hormone (FSH), decreased estrogen secretion, low anti-Müllerian hormone (AMH), and antral follicle count (AFC), which can be age dependent or independent.9 Clinicians and researchers worldwide are seeking ways to improve the number and quality of retrieved oocytes, particular for patients with DOR.
COH protocols play a critical role in determining the quantity and quality of retrieved oocytes; therefore, a proper COH protocol for patients with DOR could promote the success of IVF treatment.10 Although pituitary downregulation is the standard protocol, and yields satisfactory results for most patients, it has been demonstrated as ineffective and expensive for patients with DOR.11This is due to lengthy treatment, increased Gn use, elevating the risk of ovarian hyperstimulation syndrome (OHSS), and decreased efficacy.12,13 Recently, ovarian stimulation with clomiphene (CC), first developed by Fauser et al, has been tested in several types of patients.14–17 Commonly, CC was used to block negative feedback trigged by estrogen, thereby resulting in elevated FSH and luteinizing hormone (LH) release from the hypothalamus. Due to the short treatment-period, decreased Gn use, decreased cost, and physiological comfort, CC-primed ovarian stimulation (CPOS) is regarded as patient friendly.18,19 Furthermore, studies have demonstrated that CPOS achieves equal pregnancy outcomes on POR patients compared with the conventional COH protocol.20 Therefore, CPOS has been widely used in clinics as an ideal protocol for POR patients despite the risk of early LH peak and menstrual cycle cessation.
More recently, Kuang et al developed a new COH protocol called progestin-primed ovarian stimulation (PPOS).21 Progestin is used to block the estrogen-induced LH surge without the occurrence of OHSS.21 This protocol has been successfully used in patients with various ovarian conditions, including normal reserve and polycystic ovary syndrome.5,22,23 Kuang et al used nature cycles as a control to demonstrate that PPOS could overcome premature ovulation for patients with DOR without affecting the quality of retrieved oocytes.24 However, it is still unclear which protocol, CPOS or PPOS, is more appropriate for patients with DOR. In this study, we retrospectively analyzed data from patients with DOR who had a failed, mild ovarian stimulation and subsequently switched to the PPOS protocol. The clinical results including hormone level, oocytes retrieved, embryos fertilization and development, and clinical outcomes were compared between the two protocols that were used in the same patient.
A retrospective study was conducted at the Changzhou Maternal and Child Health Care Hospital. From January 2016 to June 2017, a total of 50 women undergoing IVF/intracytoplasmic sperm injection (ICSI) cycles due to infertility were enrolled. The study protocol was approved by the Ethics Committee of the Changzhou Maternal and Child Health Care Hospital and conducted according to the Declaration of Helsinki for medical research. All participants provided informed consent after counseling for infertility treatments and routine IVF procedures. Patients with two or more of the following items were diagnosed with DOR: (a) 25IU ≥ FSH ≥ 10IU; (b) FSH/LH ≥ 2; (c) AFC ≤ 5; and (d) AMH ≤ 0.5-1.1. Patients had to meet the following standards: (1) DOR diagnosis and (2) failure of CPOS during the last IVF cycle, resulting in a switch to PPOS. Clinical results were compared between the two treatment protocols for each patient.
2.2. Controlled ovarian stimulation
CPOS protocol: CC (50 mg/d; Cyprus Goth Pharmaceutical Co., ltd.) and Gn (150-225 IU; Anhui Fengyuan Pharmaceutical Co., China) were administered from menstrual cycle day 3 (MC3) to human chorionic gonadotropin (hCG) day. The initial dose of Gn (150-225 IU) was based on patient age, basic FSH, AFC accounts, and previous promotion plan. Ultrasound examination and serum hormone level tests were performed regularly, and the dose of Gn was adjusted according to follicle development. When the dominant follicle diameter reached >18 mm, triptorelin (0.1 mg; Decapeptyl, Ferring Pharmaceuticals) and hCG (4000 IU; Lizhu Pharmaceutical Trading Co., German) were administered to trigger ovulation. Oocytes were retrieved 36 to 38 hours later.
PPOS protocol: Medroxyprogesterone acetate (MPA; 6 mg/d; Guang Zhou Xianling Pharmaceutical Co., China) and Gn (150-225 IU; Anhui Fengyuan Pharmaceutical Co., China) were administered from MC3 onward. The remainder of the procedure was identical to CPOS.
2.3. In vitro fertilization and embryo culture
All follicles >10 mm in diameter were retrieved, and standard insemination or ICSI were performed within 6 hours depending on semen parameters. Embryos were examined for cell number, homogeneity, and the degree of embryonic fragmentation on the third day following specifications of Cummins et al. Grade I-III embryos were frozen by vitrification technology on the third day. Remaining embryos were placed in extended culture, and only blastocysts with good morphology were frozen on day 5 or 6. The cryopreservation procedure has been described previously.20 All patients in the present study had received frozen embryo transfer. Hormone replacement treatment was used for endometrial preparation. Briefly, ethinyl estrogen (25 μg tid) was administered for 14 days, and then shifted to oral progesterone (8 mg estradiol and 40 mg dydrogesterone) and soft vaginal progesterone capsules (200 mg bid; Merck Serono Co., England). Once pregnancy was achieved, exogenous estrogen and progesterone supplements were continued until 10 weeks gestation.
2.4. Laboratory analysis
Serum was collected on MC3, the day prior to, and day ovulation was triggered. Levels of FSH, LH, E2, and progesterone were measured by chemiluminescence (Abbott Biologicals B.V., The Netherlands). The lower limits of sensitivity were as follows: FSH = 0.06 mIU/mL, LH = 0.09 mIU/mL, E2 = 10 pg/mL, and P = 0.1 ng/mL.
2.5. Statistical analyses
All the acquired data were analyzed using SPSS software (SPSS version 20.0; IBM/SPSS, Inc.). The χ2 test was used to compare constituent ratio data. The paired student’s t test was used for normally distributed data, and the Wilcoxon Signed-Rank test was used for data that failed the normality test. p < 0.05 was considered statistically significant.
3.1. Basic characters
The average interval between unsuccessful IVF/ICSI with CPOS and initiation of PPOS was 117.9 ± 15.4 days, and the average infertility duration was 4.22 ± 0.61 years. The average patient age was 36.48 ± 0.83 years, BMI was 22.85 ± 0.39, basal FSH was 9.60 ± 0.71 mIU/mL, basal LH was 4.92 ± 0.58 mIU/mL, basal E2 was 26.78 ± 2.66 mIU/mL, and AFC was 3.88 ± 0.49 (Table 1).
3.2. Cycle characteristics in CPOS and PPOS
The total Gn dose was significantly lower in CPOS than PPOS treatment, while the Gn duration was similar. The LH levels were significantly higher in CPOS than PPOS treatment in the day ovulation was triggered. There were no statistical differences in FSH, E2, and P between the two treatments in the day ovulation was triggered (Table 2).
3.3. Follicle development, oocyte performance, and clinical outcomes
The number of dominant follicles (diameter >14 mm), oocytes retrieved, MII mature oocytes, normal fertilized oocytes, cleaved embryos, and high-grade embryos were significantly higher, while the miscarriage rate was significantly lower in PPOS than CPOS treatment. There were no statistical differences in cancellation rate, implantation rate, and clinical pregnancy rate between the two treatments. In addition, nine live-births were achieved after PPOS treatment, while no live-birth occurred after CPOS treatment (Table 3).
In the present study, we retrospectively compared the clinical outcomes of two COH protocols (CPOS with CC vs PPOS with MPA), which were successively used in patients with DOR. Our results showed that PPOS could significantly suppress the LH surge and yield more satisfactory results, including an increased number of dominant follicles, oocytes retrieved, MII mature oocytes, normal fertilized oocytes, cleaved embryos, high-grade embryos, number of embryos eligible for cryopreservation, live-births rate, and decreased miscarriage rate. These results are similar with a recent study comparing the outcomes from the classic long protocol and PPOS, which demonstrated the superiority of PPOS in improving oocyte utilization rate and number of high-quality embryos in aged infertile women.25
The first study reporting the use of PPOS in patients with low Gn responsiveness showed that the number of dominant follicles, oocytes retrieved, MII mature oocytes, normal fertilized oocytes, viable embryos, clinical pregnancy rate, implantation rate, and live-birth rate were higher, while miscarriage rate was lower compared to patients in a natural cycle. However, the difference of clinical pregnancy rate, implantation rate, live-birth rate, and miscarriage rate have no statistical significance.24 Although the differences in clinical pregnancy rate and implantation rate between the two treatments were not significant in our study, we found that miscarriage and live-birth rates were significantly different, indicating that the improved oocyte quality achieved following PPOS contribute to a live-birth in patients with DOR. A self-controlled study with fewer variables may draw a confirmative conclusion with limited samples. Therefore, our results highlight the importance of PPOS in improving folliculogenesis or the quality of oocytes.
The possible mechanisms underlying the inhibitory effect of MPA on the early LH surge were explained by Kuang et al.21 Clinical trials have demonstrated that, in the presence of a low level of E2, P administration could significantly inhibit LH surge.21 It is known that the progestin receptor (PR) is crucial for the E2-induced LH surge.26,27 E2 can induce PR expression in the hypothalamus, while progestin downregulates its own receptor.28 High levels of progestin in the absence of adequate E2 will maintain a relatively low level of PR, thereby abolishing the E2 positive feedback pathway, resulting in low LH release.
However, the mechanisms by which MPA improves the number and quality of oocytes remain uncharacterized. Kuang et al proposed that administration of human menopausal gonadotropin (hMG) during the late follicular phase of the MPA treatment may contribute to the higher number of retrieved oocytes and embryos.24 In the present study, hMG was used in both treatments and we noted that a higher dose was used in PPOS than CPOS treatment. However, the FSH level in the trigger day between the two treatments was similar. Several studies have demonstrated that increased Gn use did not benefit patients with DOR, especially those with low AFC.11,29 Thus, it was not likely that the improved number and quality of oocytes retrieved can be attributed to increased Gn use in PPOS treatment.
LH plays a critical role in synthesizing and secreting androgens, which are required for further production of E2. It has been demonstrated that low androgen levels promote folliculogenesis, while high levels inhibit folliculogenesis.30,31This suggests that LH levels should be maintained in a suitable range. Extremely low or high LH levels will have negative influences on the outcomes of IVF/ICSI.32,33 In the present study, no premature LH surge occurred in either treatment. In addition, E2 levels were similar at the time of ovulation, indicating that a high level of LH in CPOS treatment did not further promote E2 synthesis. A recently published article showed that CC increased LH levels during PPOS treatment without affecting IVF/ICSI outcomes, indicating that oocyte improvements associated with PPOS are likely unrelated to LH levels.34 A recent publication showed differentially elevated lipids in follicular fluid between PPOS and a short treatment protocol, and that difference may be associated with improved IVF/ICSI outcomes.35 Future studies are needed to investigate the exact mechanisms regarding how MPA improves folliculogenesis.
In conclusion, our before-and-after self-controlled study demonstrated that PPOS could not only suppress the LH surge but also improve the quantity and quality of oocytes through improved folliculogenesis in patients with DOR, suggesting that PPOS treatment is an ideal ovarian stimulation protocol for patients with DOR.
This study was supported by a basic and applied research project from Changzhou (CJ20179049) and a special fund for clinical research from the Chinese Medical Association (17020490718).
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