1. Introduction
Poor ovarian response (POR) refers to the reduction in ovarian reserve and the insensitivity of ovaries to gonadotropins (Gn) during ovarian stimulation therapy in infertile patients. POR has always been a challenge for in vitro fertilization and embryo transfer (IVF-ET) technology because the number of oocytes obtained after controlled ovarian stimulation (COS) will decrease. The incidence of POR in the infertile population is about 9% to 24% [
1], which is characterized by decreased ovarian reserve indicators such as anti-Müllerian hormone (AMH), insensitivity to ovarian stimulation drugs, and difficulty in obtaining sufficient mature oocytes, etc. In patients with prospective POR, a high dose of Gn did not significantly improve the number of oocytes retrieved and the number of available embryos, let alone pregnancy outcomes [
2]. Thus, the mild stimulation protocol with low-dose Gn and an estrogen antagonist (such as clomiphene) as adjuvants seems to be a cost-effective treatment option for POR patients [
3]. In recent years, a new COS protocol called the double stimulation (DouStim) protocol has been developed and applied in POR patients. That is, in the same menstrual cycle, two consecutive ovarian stimulations and oocyte retrievals are performed in the follicular phase and the luteal phase, respectively. DouStim was first proposed by Kuang et al. in 2014, also known as the “Shanghai Protocol” [
4]. Theoretically, ovarian follicles only develop during the “follicular phase”. In fact, a previous study has shown that there are two or three waves of follicular development in a menstrual cycle [
5]. In addition to the biphasic stimulation regimen based on the mild stimulation protocol proposed by Kuang, some physicians have used a dual ovarian stimulation regimen based on a pituitary suppression protocol. Recruitment and collection of luteal phase follicles has been demonstrated to be feasible [
6]. Unlike the traditional mild stimulation protocol where ovarian stimulation and oocyte retrieval are performed only once during the follicular phase, the DouStim protocol involves conducting it twice in one menstrual cycle. Therefore, it is inappropriate to compare clinical indicators and pregnancy outcomes between DouStim and one single mild stimulation cycle; it is more accurate to compare the DouStim protocol with the protocol of two consecutive mild stimulations. However, there is little research in this area. This retrospective study took POR patients who underwent DouStim as study objects and those who underwent two consecutive mild stimulations as controls to compare the differences in laboratory parameters and pregnancy outcomes and to explore risk factors and protective factors that may affect pregnancy outcomes.
4. Discussion
Our study population is not completely consistent with the POSEIDON classification, so the Bologna criteria were adopted as the diagnostic standard for POR. Previous studies have shown that POR patients who underwent DouStim in “one menstrual cycle” harvested more oocytes and usable embryos. This may improve the CLBR in patients diagnosed as “expected POR” [
9]. DouStim involves two oocyte retrieval operations, so some scholars have questioned whether there is a difference between complete DouStim and two conventional ovarian stimulations. It still needs further investigation [
10]. Therefore, the purpose of this study was to compare DouStim and the two consecutive mild stimulations protocol in the POR population. Due to the high incidence of aneuploid embryos in elderly patients [
11], improvement of the ovulation stimulation protocol alone often has no significant effect on pregnancy outcomes. Therefore, this study did not include elderly patients over 40 years old with POR. Overall, the study population can be categorized as POSEIDON groups 3 and 4, except for patients over 40 years old.
This study showed that in the two groups of POR patients with the same baseline indicators, the duration and total dose of Gn in the DouStim group were higher than those in the two consecutive mild stimulations group. The physiological characteristic of lower levels of endogenous gonadotropins in the luteal phase makes the LPS need a higher initial dose of Gn (225 U/d) and a longer duration [
12]. The endocrine conditions (such as LH, E
2, and P
4) on the trigger day were also different; for example, the serum P4 level on the trigger day was significantly higher in the DouStim group, which was related to the luteal phase. In addition, while there was no difference in the number of oocytes retrieved between the two groups, the number of frozen embryos in the DouStim group was lower than that in the two consecutive mild stimulations group, and the proportion of patients without frozen embryos was higher, which means that the efficiency of the DouStim protocol in obtaining available embryos was lower than that of the two consecutive mild stimulations protocol. In 2014, Kuang et al. reported for the first time that 38 POR patients had undergone dual ovarian stimulation. The number of high-quality embryos and frozen embryos and the cleavage rate of fertilized oocytes obtained showed no difference between the double phases. However, the number of oocytes and usable embryos obtained in LPS was more than that in FPS. Therefore, they believed that DouStim could improve pregnancy outcomes [
4]. Vaiarell and Jin argued that POR patients undergoing the DouStim regimen may improve their pregnancy outcomes because of the relatively high oocyte production level of LPS [
13,
14]. The above conclusions were based on the comparison of pregnancy outcomes between the DouStim protocol and one single COS cycle. Our study demonstrated that, compared with the two continuous mild stimulations protocol, the DouStim protocol could not significantly increase the production of oocytes and embryos even if the duration and dose of Gn were amplified in POR patients.
In this study, in the case of similar basic indicators such as the number of frozen embryos transferred and the number of FETs, there was no difference between the two groups in terms of pregnancy outcomes, including CPR and CLBR, as well as pregnancy complications such as early abortion rate, ectopic pregnancy rate, premature birth rate, and birth defect rate. Recently, Lu’s meta-analysis summarized recent studies on LPS, including 4433 patients. The results showed that there were similar results in pregnancy outcomes, indicating that LPS was not inferior to traditional follicular stimulation [
15]. Yet the evidence for the wide application of LPS is insufficient. Cimadomo and Ubaldi et al. studied LPS and FPS at the genetic level and concluded that there was no significant difference in the gene expression of oocyte quality and in the follicular microenvironment [
16,
17]. Other experimental studies have shown that LPS-stimulated cumulus cells differ from traditional ovarian stimulation in gene expression (inflammation, oxidative phosphorylation, and apoptosis), which may have a negative impact on the mitochondrial function and immune response of oocytes and increase oocyte apoptosis and abnormal glucose metabolism. However, there was no significant difference between them in clinical observation indexes and pregnancy outcomes [
18]. Chen et al. conducted a retrospective study on LPS-induced pregnancy and delivery and found no significant difference in congenital malformation rate, neonatal defect rate, and neonatal weight [
19]. These studies and our study have confirmed that LPS will not damage pregnancy outcomes. The birth defect rate was 12% in the DouStim group and 5.9% in the two consecutive mild stimulations group, but there was no statistical difference (
p > 0.05). We considered that the high value of the DouStim group was due to the small number of live birth samples. Therefore, if there are two or more antral follicles with a diameter of less than 8mm in the bilateral ovaries of POR patients on the trigger day of FPS, biphasic stimulation is still a feasible option.
Depending on univariate analysis or clinical practice, several variables were included in the multivariate logistic regression for further analysis. This study found that age was a risk factor for pregnancy outcomes, while different COS protocols and other baseline data had no impact. This indicates that no matter what stimulation protocol is used, the pregnancy outcomes of elderly patients with POR will be worse. Further subgroup comparison based on age showed that there was no difference in clinical observation indicators and pregnancy outcomes between the two stimulation protocols in the young POR group. Although there was no significant difference in the yield of oocytes [
20], the number of frozen embryos in the DouStim group was significantly lower than that in the two consecutive mild stimulations group in the elderly POR group (ages ≥ 35 years). This result indicates that the differences between the two stimulation protocols mainly occurred in the elderly subgroup.
There are some limitations in this study. As a retrospective study with a relatively small sample size, this study inevitably has selection bias. Furthermore, due to the limitation of the observation time window and patient selection, the pregnancy outcomes after all embryo transfers could not be observed.