To the best of our knowledge, this is the first study to investigate different mRNA expression in human CCs between LPOS and FPOS. This prospective study suggested that ovarian stimulation started from luteal phase or follicular phase could influence CCs mRNA expression which are related to inflammation, oxidative-phosphorylation, apoptosis and metabolism. However, in this study, the number of retrieved oocytes, metaphase II oocytes, embryos on Day 3, top-quality embryos on Day 3, clinical pregnancy rate and live birth rate were not significantly different between LPOS and FPOS, mainly due to small population.
This study demonstrated that lower mRNA levels of CXCL1 and PTGES was found in the LPOS group than in the FPOS group. CXCL1 (C-X-C motif chemokine ligand 1), also called interleukin 1 (IL-1), is a member of the CXC subfamily of chemokines. CXCL1 plays a role in inflammation and as a chemoattractant for neutrophils. A prospective study conducted by Zollnerand colleagues enrolled 256 couples undergoing the IVF/ICSI cycles, showing that high levels of IL-1 beta in the follicular fluid were positively associated with fertilization rates (23). Furthermore, a prospective study of Rehman et al. including a total of 323 patients opting for ICSI demonstrated that higher serum IL-l beta levels were observed in clinical pregnancy group than non-pregnant group or preclinical abortion group (24). PTGES (prostaglandin E synthase) has three known forms: PTGES1, PTGES2, and PTGES3 (25). PTGES is a key enzyme required for the synthesis of PGE2, specifically converting PGH2 to PGE2 (26). During the maturation process of bovine oocytes, PTGES, especially PTGES1, works in coordination with PTGS2 to stimulate PGE2 generation (27). During the process of oocyte maturation, PGE2 plays a vital role in cumulus expansion and oocyte meiosis resumption (28). In addition to oocyte maturation, PGE2 has been showed to be a critical mediator to promote successful fertilization, embryo development and early implantation (29).
The mRNA expression of NDUFB7 and NDUFA4L2 was significantly lower in the LPOS group compared with the FPOS group in this study. NDUFB7 (NADH:ubiquinone oxidoreductase subunit B7) and NDUFA4L2 (NDUFA4 mitochondrial complex associated like 2) encode the protein involved in the electron transport chain which is the main process of ATP production in the mitochondria. Numerous studies have indicated that mitochondrial dysfunction of oocytes has the negative impact on oocyte maturation, fertilization, embryo development, and pregnancy (30–32). Additionally, this study showed that the LPOS group presented higher mRNA expression of DAPK3 and BCL6B than the FPOS group. DAPK3 (death-associated protein kinase 3) and BCL6B (BCL6B transcription repressor) both play a role in the induction of apoptosis. Increased apoptosis of CCs has been reported to be poorly associated with oocyte maturation, fertilization, embryo development, and pregnancy (33–35). Accordingly, this study seemed to reveal that LPOS may lead to mitochondrial dysfunction and increased apoptosis of CCs, causing adverse reproductive outcomes.
Moreover, in this study, increased mRNA expression of PCK1 and decreased mRNA expression of LDHC were observed in the LPOS group compared to the FPOS group. PCK1 (phosphoenolpyruvate carboxykinase 1) is a central regulator of gluconeogenesis and is regulated by Cited2. Fang et al. demonstrated that the high Cited2 protein levels in CCs significantly increased the expression of PCK1 mRNA and glucose in CCs. It was suggested that the high Cited2 level might impair oocyte quality via up-regulating PCK1 mRNA expression to result in abnormal glucose metabolism in CCs (36). Therefore, increased mRNA expression of PCK1 in the LPOS group seemed to have unfavorable influence on oocytes by disordering glucose metabolism. LDHC (lactate dehydrogenase C) catalyzes the conversion of L-lactate and NAD to pyruvate and NADH in the final step of anaerobic glycolysis. Enhanced glucose metabolism by increasing activity of glycolysis in oocytes was positively associated with oocyte maturation and embryo development (37–39). Hence, LPOS seemed to harm oocyte competence and embryo growth by decreasing LDHC mRNA expression.
Taken together, the present study showed that LPOS may diminish IL-1 and PGE2 production, reduced mitochondrial function and elevate apoptosis, increase gluconeogenesis and decrease glycolysis in the CCs, implying that LPOS might have detrimental effects on the CCs. Although it has been proposed that PORs may earn benefit from LPOS because physiologic elevated progesterone could prevent premature LH rise naturally in the luteal phase (7), there were no definite clinical evidence to support it. Some studies revealed that the LPOS increased the chance to gain more competent oocytes and embryos in PORs compared with the FPOS (7–9). However, these studies were not randomized controlled trials and had small numbers of patients. The only randomized controlled pilot trial performed by Kansal Kalra and colleagues revealed that IVF outcomes, including number of oocytes retrieved and embryos transferred, clinical pregnancy rate and live birth rate, between LPOS and FPOS were similar in PORs (10). Additionally, a retrospective study conducted by Wu et al. included 274 PORs, suggesting that there was no significant difference regarding mean number of retrieved oocytes and embryos, implantation and clinical pregnancy rates between LPOS and FPOS (11), the same results as the current study in spite of quite small population. Admittedly, progestins have been proven to be able to inhibit early-onset LH surge effectively, but the effects of high levels of progesterone on oocytes or CCs were still unclear. This study showed that LPOS might have harmful effects on CCs. However, large-scare randomized controlled trials are required to confirm the results from this study.
Several limitations of our study should be taken into account for data interpretation. First, this was a non-randomized trial and had small study population. Second, the participants enrolled based on Bologna criteria may be heterogeneous. Third, limited CCs genes were analyzed in this study. However, the strength of this study was that all the IVF protocols were carried out by the same physician and all the laboratory procedures were executed by the same embryologist, which minimize the bias in performance.
In conclusion, this study showed LPOS might have disadvantageous influence on CCs via decreased expression of CXCL1, PTGES, NDUFB7, NDUFA4L2, LDHC and increased expression of DAPK3, BCL6B, PCK1, indicating that LPOS seemed to diminish beneficial inflammation and mitochondrial function, and augment apoptosis and abnormal glucose metabolism in CCs. However, further randomized controlled trials with large populations are needed to verify these results.