Luteal phase ovarian stimulation versus follicular phase ovarian stimulation results in different human cumulus cells gene expression: a pilot study

Luteal phase ovarian stimulation versus follicular phase ovarian stimulation results in different human cumulus cells gene expression: a pilot study | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Luteal phase ovarian stimulation versus follicular phase ovarian stimulation results in different human cumulus cells gene expression: a pilot study Li-Te Lin, Ju-Yueh Li, Kuan-Hao Tsui, Chia-Jung Li, Peng-Hui Wang, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.2.22377/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract OBJECTIVE: Physiologic elevated levels of progesterone in luteal phase can impede early-onset LH surge. However, the impact of high levels of progesterone on the oocyte or cumulus cells (CCs) remains indistinct. Therefore, the aim of study was to investigate the CCs gene expression between luteal phase ovarian stimulation (LPOS) and follicular phase ovarian stimulation (FPOS) in poor ovarian responders (PORs) undergoing in vitro fertilization (IVF) cycles. MATERIALS AND METHODS: This was a prospective non-randomized trial (ClinicalTrials.gov Identifier: NCT03238833). A total of 36 PORs who conformed Bologna criteria and underwent IVF cycles were enrolled. 15 PORs were allocated to the LPOS group and 21 PORs were allocated to the FPOS group. Basic characteristics, cycle characteristics and pregnancy outcomes were compared between the two groups. Moreover, CCs genes regarding inflammation (CXCL1, CXCL3, TNF, PTGES), oxidative-phosphorylation (NDUFB7, NDUFA4L2, SLC25A27), apoptosis (DAPK3, BCL6B) and metabolism (PCK1, LDHC) were analyzed using real-time quantitative PCR between the two groups. RESULTS: Basic characteristics and IVF outcomes were similar between the two groups except significantly high progesterone level in the LPOS group. The mRNA expression of CXCL1 and PTGES were significantly lower in the LPOS group than in the FPOS group ( p < 0.05). The LPOS group had significantly lower mRNA expression of NDUFB7 and NDUFA4L2 than the FPOS group ( p < 0.05). DAPK3 and BCL6B mRNA expression were significantly higher in the LPOS group compared to FPOS group ( p < 0.05). Increased expression of PCK1 and decreased expression of LDHC were observed in the LPOS group compared to the FPOS group. ( p < 0.05). CONCLUSIONS: Compared to the FPOS, the LPOS seemed to reduce favorable inflammation and mitochondrial function, and induce apoptosis and abnormal glucose metabolism in CCs. Cancer Biology Sexual & Reproductive Medicine cumulus cells follicular phase ovarian stimulation gene expression luteal phase ovarian stimulation poor ovarian responders Figures Figure 1 Figure 2 Figure 3 Introduction Luteal phase ovarian stimulation (LPOS), referring to the initiation of ovarian stimulation from the luteal phase, has been regarded as a feasible protocol for in vitro fertilization (IVF) cycles (1) following the theory of multiple follicular recruitment waves in the same menstrual cycle had been proposed (2). LPOS was first applied in the fertility preservation of cancer patients (3, 4), and then used in the general infertile couples (5, 6). Studies showed that similar number of retrieved oocytes and mature oocytes and fertilization rate were noted between LPOS and follicular phase ovarian stimulation (FPOS) in the urgent fertility preservation (3, 4) or women with normal ovarian response (5, 6). In poor ovarian responders (PORs), some studies revealed that more competent oocytes and embryos could be obtained in the LPOS, compared to FPOS (7–9). The possible rationale was that physiologic high levels of progesterone in the luteal phase could effectively block premature luteinizing hormone (LH) surge which more frequently occurred in the PORs during ovarian stimulation. Our previous study demonstrated that the numbers of retrieved oocytes, metaphase II oocytes, fertilized oocytes, and day-3 embryos were significantly higher in the LPOS group than in the FPOS group (7). However, some studies had conflicting results (10, 11). Furthermore, there was lack of large-scare randomized controlled trials to bolster the consequences. Therefore, no solid evidence support that PORs could really get more benefit from LPOS until now. Cumulus cells (CCs) are somatic cells surrounding the oocyte in cumulus-oocyte complexes (COCs). Bidirectional intercellular communication between CCs and the oocyte mediated by a network of specialized gap junctions is crucial for development of follicles (12). Oocyte-secreted factors, such as growth-differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15), generated from the oocyte regulate proliferation, apoptosis, luteinization, metabolism and expansion of CCs (13). CCs protect and nurture the oocyte, playing an essential role in oocyte maturation, ovulation and fertilization (14). Therefore, the expression profiles of CCs have the potential to reflect oocyte competence and even serve as predictors to determine embryo quality, pregnancy and live birth outcomes (15–17). Although progesterone can prevent premature luteinization effectively (18, 19), the influence of high levels of progesterone on oocytes or CCs were poorly understood. Therefore, the goal of this study was to investigate the differences of human CCs gene expression between LPOS and FPOS. Materials And Methods Study Population and design This prospective cohort study was implemented at the Reproductive Medicine Center of the Kaohsiung Veterans General Hospital from August 2017 to December 2018. We enrolled PORs undergoing IVF cycles. The inclusion criteria for POR in this study were defined according to the Bologna Criteria (20), having at least two of the three following features: (i) advanced maternal age (≥ 40 years) or any other risk factor for POR; (ii) a previous POR (≤ 3 oocytes with a conventional stimulation protocol); (iii) an abnormal ovarian reserve test. An abnormal ovarian reserve test was defined as antral follicle count (AFC) < 5 or anti-Müllerian hormone (AMH) < 1 ng/mL in this study. Furthermore, two episodes of a previous POR after maximal stimulation alone would be sufficient to define a patient as a POR. Patients were excluded if they had any of the following: (i) a diagnosis of primary ovarian insufficiency; (ii) a history of oophorectomy; (iii) a history of exposure to cytotoxic agents or pelvic irradiation for malignancy; (iv) a history of adjuvant supplementation or hormonal replacement therapy during the previous 3 months. The enrolled participants were then divided into two groups: follicular phase ovarian stimulation (FPOS) or luteal phase ovarian stimulation (LPOS). The choice of ovarian stimulation protocol was determined by physicians’ consideration. Baseline and cycle characteristics and IVF outcomes were compared between the two groups. Ethics Statement This study was approved by the institutional review board of Kaohsiung Veterans General (VGHKS15-CT11-12) and Clinical Trial Register (ClinicalTrials.gov Identifier: NCT03238833). All participants were fully counselled and written informed consent was obtained. This study was performed adherence to approved guidelines and the Declaration of Helsinki. Treatment protocol In FPOS group, ovarian stimulation with a 300 IU daily dose of combined recombinant follicle stimulating hormone (rFSH) plus recombinant LH (rLH) (Pergoveris, Merck Serono, Aubonne, Switzerland) was commenced within 5 days of the menstrual cycle. In LPOS group, spontaneous ovulation was confirmed by transvaginal sonography and progesterone level from day 15 to day 18 of the menstrual cycle. After confirmation of spontaneous ovulation, the women with at least one follicle of less than 8 mm started to undergo ovarian stimulation with a 300 IU daily dose of rFSH plus rLH (Pergoveris, Merck Serono, Aubonne, Switzerland). In both FPOS and LPOS groups, when the leading follicle reached 12 mm in diameter, the women received 0.25 mg of GnRH antagonist (Cetrotide; Merck Serono, Idron, France) daily until the day of oocyte trigger. Dual trigger, combination of recombinant human chorionic gonadotropin (rHCG) (Ovidrel, Merck Serono, Modugno, Italy) and GnRH agonist (Lupro, Nang Kuang Pharmaceutical Co., Ltd., Tainan, Taiwan), was administered when at least one dominant follicle reached the size of 17 mm. 36 hours after ovulation induction, oocyte retrieval was conducted by transvaginal ultrasound-guided needle aspiration. Oocytes were inseminated by intracytoplasmic sperm injection (ICSI) for all women in order to diminish the possibility of fertilization failure. Oocytes were denuded and inseminated if maturation status was verified by the presence of the first polar body. Fertilization was evaluated 18 ~ 20 hours after insemination and was defined success by the presence of two pronuclei. Embryos development and quality were assessed based on the number, symmetry of the blastomeres and embryonic fragmentation according to the criteria established by the Istanbul consensus workshop (21). All embryos were cryopreserved by vitrification on the third day after oocyte retrieval. An artificial frozen embryo transfer protocol was used for all participants. Oral estradiol (Ediol 8 mg, Synmosa Biopharma Corporation, Hsinchu County, Taiwan) was initiated on the third day of the menstrual cycle and endometrial thickness was monitored by transvaginal ultrasonography. When the endometrial thickness exceeded 8 mm, luteal support with progesterone intravaginal gel (Crinone 8% gel 90 mg/day, Merck Serono, Hertfordshire, UK) plus oral dydrogesterone (Duphaston 40 mg, Abbott, Olst, The Newtherlands) was added. Transabdominal ultrasound-guided embryo transplantation was performed 4 days after commencement of luteal support. The women underwent a pregnancy test 15 days after embryo transfer. If the pregnancy test was positive, progesterone was continued until 8 ~ 10 weeks of gestation. Clinical pregnancy was defined by the presence of fetal cardiac activity in an intrauterine gestational sac by transvaginal ultrasound. Live birth was determined by delivery of a live fetus after 20 weeks of gestation. Cumulus cells collection and genes expression COCs were collected during oocyte aspiration and washed in the medium. CCs were removed mechanically using a sterile scalpel. CCs separated from the same patient’s COCs were pooled together for study. Isolated CCs were then transferred immediately into a sterile tube, centrifuged at 200 g for 5 min at room temperature and stored at -80 °C for further study. CCs were analyzed for the expression of genes related to inflammation (CXCL1, CXCL3, TNF, PTGES), oxidative-phosphorylation (NDUFB7, NDUFA4L2, SLC25A27), apoptosis (DAPK3, BCL6B) and metabolism (PCK1, LDHC) using a real-time quantitative reverse-transcription polymerase chain reaction (qRT-PCR). RNA extraction and real-time qRT-PCR As previously described (22), total RNA was extracted from CCs with the use of the TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. Each RNA pool was reverse transcribed to cDNA. To detect mRNA expression, real-time qRT-PCR analysis was performed using an ABI Prism 7700 Sequence Detection System (Perkin-Elmer Applied Biosystems, Foster City, CA, USA). PCR was performed using the SYBR Green PCR Core Reagents kit (Perkin-Elmer Applied Biosystems). Gene-specific qRT-PCR primers that were used are shown in supplemental Table S1. The thermal cycling conditions included an initial denaturation step at 95 °C for 10 min, 40 cycles at 95 °C for 15 s, and 60 °C for 1 min. Each set of qRT-PCR reactions was repeated three times. All of the samples with a coefficient of variation for Ct value > 1% were retested. The GAPDH served as an internal control to normalize the expression of target genes. Relative expression levels were calculated for each sample after normalization against GAPDH. Statistical analysis The statistical analysis was carried out using the Statistical Package for Social Sciences (SPSS) version 22.0 (Chicago, IL, USA). The 2-tailed Student’s t-test were used to compare quantitative variables. The categorical variables were compared using Chi-Square tests or Fisher’s exact tests. The differences between groups were considered significant when the p value was less than 0.05. Results Comparison of basic characteristics between FPOS and LPOS groups A total of 36 patients were recruited in this study and divided into FPOS (n = 21) and LPOS (n = 15) groups. The baseline characteristics in the two groups are presented in Table 1 . The mean age (39.7 ± 3.8 years vs. 40.0 ± 3.4 years) and body mass index (21.7 ± 3.1 kg/m 2 vs. 23.6 ± 3.8 kg/m 2 ) of patients between the two groups were similar. Additionally, there were no statistically significant differences between groups in terms of infertility duration, previous IVF attempts, primary or secondary infertility, basal FSH, AFC and AMH. Table 1 Basic characteristics of poor ovarian responders undergoing follicular phase ovarian stimulation (FPOS) or luteal phase ovarian stimulation (LPOS) Parameters FPOS (n = 21) LPOS (n = 15) p value Age (years) 39.7 ± 3.8 40.0 ± 3.4 0.818 Body mass index (kg/m2) 21.7 ± 3.1 23.6 ± 3.8 0.128 Infertility duration (years) 5.1 ± 3.2 5.3 ± 6.6 0.882 Previous IVF attempts (n) 2.9 ± 2.9 2.1 ± 2.6 0.441 Types of infertility (%) 0.204 Primary infertility 52.4 73.3 Secondary infertility 47.6 26.7 Basal FSH (IU/l) 5.6 ± 4.0 5.4 ± 2.2 0.864 Antral follicle counts (n) 4.4 ± 1.4 4.9 ± 1.7 0.273 Anti-Müllerian hormone (ng/ml) 0.7 ± 0.4 0.7 ± 0.6 0.441 Data are presented as mean ± standard deviation or percentage. IVF, in vitro fertilization; FSH, follicle stimulation hormone Comparison of cycle characteristics and pregnancy outcome between FPOS and LPOS groups The stimulation cycle outcomes of each group are shown in Table 2 . No statistically significant difference existed in duration of stimulation, total dose of gonadotrophins, or peak serum estradiol level. However, the peak serum progesterone level was significantly higher in LPOS group than FPOS group (6.8 ± 6.8 vs. 0.5 ± 0.2, p = 0.004). Table 2 Cycle characteristics and pregnancy outcome of poor ovarian responders undergoing follicular phase ovarian stimulation (FPOS) or luteal phase ovarian stimulation (LPOS) Parameters FPOS (n = 21) LPOS (n = 15) p value Stimulation duration (days) 11.3 ± 2.2 12.1 ± 2.8 0.363 Gonadotropin dosage (IU) 2882.1 ± 690.1 2885.0 ± 744.0 0.991 Peak estradiol (pg/mL) 749.9 ± 553.2 756.7 ± 671.5 0.975 Progesterone (ng/ mL) 0.5 ± 0.2 6.8 ± 6.8 0.004 No. of oocytes retrieved (n) 3.0 ± 1.4 3.1 ± 1.6 0.713 No. of metaphase II oocytes (n) 2.2 ± 1.5 2.3 ± 1.3 0.847 Maturation rate (%) 69.1 ± 36.7 72.9 ± 28.5 0.742 No. of fertilized oocytes (n) 1.4 ± 1.1 2.0 ± 1.3 0.207 Fertilization rate (%) 61.7 ± 41.1 79.6 ± 32.2 0.170 No. of Day 3 embryos (n) 1.5 ± 1.2 1.9 ± 1.3 0.357 No. of top-quality Day 3 embryos (n) 0.4 ± 0.6 0.7 ± 1.0 0.249 Clinical pregnancy rate (%) 14.3 13.3 0.935 Live birth rate (%) 9.5 13.3 0.720 Data are presented as mean ± standard deviation or percentage. No difference was observed between FPOS and LPOS groups regarding the number of retrieved oocytes (3.0 ± 1.4 vs. 3.1 ± 1.6, p = 0.713), metaphase II oocytes (2.2 ± 1.5 vs. 2.3 ± 1.3, p = 0.847), fertilized oocytes (1.4 ± 1.1 vs. 2.0 ± 1.3, p = 0.207), embryos on Day 3 (1.5 ± 1.2 vs. 1.9 ± 1.3, p = 0.357) and top-quality embryos on Day 3 (0.4 ± 0.6 vs. 0.7 ± 1.0, p = 0.249). Moreover, the clinical pregnancy rate (14.3% vs. 13.3%, p = 0.935) and live birth rate (9.5% vs. 13.3%, p = 0.720) were similar between the two groups. Cumulus cells gene expression between FPOS and LPOS groups As shown in the Fig. 1 , regarding inflammation related genes, CXCL1 (0.51 vs 1.00, p < 0.001) and PTGES (0.30 vs 1.00, p < 0.01) mRNA expression was significantly lower in the LPOS group than in the FPOS group. However, mRNA expression of CXCL3 and TNF were not significantly different between the two groups. In the Fig. 2 , regarding genes related to oxidative-phosphorylation, NDUFB7 (0.12 vs 1.00, p < 0.001) and NDUFA4L2 (0.33 vs 1.00, p < 0.01) were expressed at lower levels in the LPOS group than in the FPOS group. However, the mRNA expression of SLC25A27 was similar between the two groups. In terms of apoptosis related genes, DAPK3 (3.81 vs 1.00, p < 0.05) and BCL6B (2.59 vs 1.00, p < 0.01) were more highly expressed in the LPOS group, compared to the FPOS group. In terms of metabolism related genes (Fig. 3 ), compared to the FPOS group, significant increased PCK1 mRNA expression (3.13 vs 1.00, p < 0.001) and decreased LDHC mRNA expression (0.12 vs 1.00, p < 0.001) were found in the LPOS group. Discussion 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. List of abbreviations AFC: antral follicle count; AMH: anti-Müllerian hormone; BCL6B: BCL6B transcription repressor; BMP15: bone morphogenetic protein 15; CC: cumulus cell; COC: cumulus–oocyte–complex; CXCL1: C-X-C motif chemokine ligand 1; DAPK3: death-associated protein kinase 3; FPOS: follicular phase ovarian stimulation; FSH: follicle stimulation hormone; GDF9: growth-differentiation factor 9; ICSI: intracytoplasmic sperm injection; IL-1: interleukin 1; IVF: in vitro fertilization; LDHC: lactate dehydrogenase C; LH: luteinizing hormone; LPOS: Luteal phase ovarian stimulation; NDUFA4L2: NDUFA4 mitochondrial complex associated like 2; NDUFB7:NADH:ubiquinone oxidoreductase subunit B7; PCK1: phosphoenolpyruvate carboxykinase 1; POR: poor ovarian responder; PTGES: prostaglandin E synthase; qRT-PCR: real-time quantitative reverse-transcription polymerase chain reaction; rFSH: recombinant follicle stimulating hormone; rHCG: recombinant human chorionic gonadotropin Declarations Ethics approval and consent to participate The study conformed to the ‘‘Declaration of Helsinki for Medical Research involving Human Subjects’’. Additionally, approval was obtained from the institutional review board at Kaohsiung Veterans General Hospital, with the identifier VGHKS15-CT11-12. The study was performed in accordance with approved guidelines. Consent for publication Not applicable. Availability of data and materials The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. Competing interests The authors declare that they have no competing interests. Funding disclosure No funding. Authors' contributions PH and ZH contributed conception and design of the study; LT and CJ organized the database and performed the statistical analysis; CJ did the laboratory research; LT and JY wrote the first draft of the manuscript; LT, JY and KH wrote sections of the manuscript. All authors contributed to manuscript revision, read and approved the submitted version. Acknowledgements This work was generously supported by grants VGHNSU107-013 from Kaohsiung Veterans General Hospital. We thank the Economy Company Ltd. for their technical and English editing service. References Kuang Y, Hong Q, Chen Q, Lyu Q, Ai A, Fu Y, et al. Luteal-phase ovarian stimulation is feasible for producing competent oocytes in women undergoing in vitro fertilization/intracytoplasmic sperm injection treatment, with optimal pregnancy outcomes in frozen-thawed embryo transfer cycles. Fertility and sterility. 2014;101(1):105-11. Baerwald AR, Adams GP, Pierson RA. Ovarian antral folliculogenesis during the human menstrual cycle: a review. Human reproduction update. 2012;18(1):73-91. Cakmak H, Katz A, Cedars MI, Rosen MP. Effective method for emergency fertility preservation: random-start controlled ovarian stimulation. Fertility and sterility. 2013;100(6):1673-80. von Wolff M, Thaler CJ, Frambach T, Zeeb C, Lawrenz B, Popovici RM, et al. Ovarian stimulation to cryopreserve fertilized oocytes in cancer patients can be started in the luteal phase. Fertility and sterility. 2009;92(4):1360-5. Martinez F, Clua E, Devesa M, Rodriguez I, Arroyo G, Gonzalez C, et al. Comparison of starting ovarian stimulation on day 2 versus day 15 of the menstrual cycle in the same oocyte donor and pregnancy rates among the corresponding recipients of vitrified oocytes. Fertility and sterility. 2014;102(5):1307-11. Qin N, Chen Q, Hong Q, Cai R, Gao H, Wang Y, et al. Flexibility in starting ovarian stimulation at different phases of the menstrual cycle for treatment of infertile women with the use of in vitro fertilization or intracytoplasmic sperm injection. Fertility and sterility. 2016;106(2):334-41 e1. Lin LT, Vitale SG, Chen SN, Wen ZH, Tsai HW, Chern CU, et al. Luteal Phase Ovarian Stimulation May Improve Oocyte Retrieval and Oocyte Quality in Poor Ovarian Responders Undergoing In Vitro Fertilization: Preliminary Results from a Single-Center Prospective Pilot Study. Adv Ther. 2018;35(6):847-56. Wei LH, Ma WH, Tang N, Wei JH. Luteal-phase ovarian stimulation is a feasible method for poor ovarian responders undergoing in vitro fertilization/intracytoplasmic sperm injection-embryo transfer treatment compared to a GnRH antagonist protocol: A retrospective study. Taiwanese journal of obstetrics & gynecology. 2016;55(1):50-4. Li Y, Yang W, Chen X, Li L, Zhang Q, Yang D. Comparison between follicular stimulation and luteal stimulation protocols with clomiphene and HMG in women with poor ovarian response. Gynecological endocrinology : the official journal of the International Society of Gynecological Endocrinology. 2016;32(1):74-7. Kansal Kalra S, Ratcliffe S, Gracia CR, Martino L, Coutifaris C, Barnhart KT. Randomized controlled pilot trial of luteal phase recombinant FSH stimulation in poor responders. Reproductive biomedicine online. 2008;17(6):745-50. Wu Y, Zhao FC, Sun Y, Liu PS. Luteal-phase protocol in poor ovarian response: a comparative study with an antagonist protocol. J Int Med Res. 2017:300060516669898. Zuccotti M, Merico V, Cecconi S, Redi CA, Garagna S. What does it take to make a developmentally competent mammalian egg? Human reproduction update. 2011;17(4):525-40. Gilchrist RB, Lane M, Thompson JG. Oocyte-secreted factors: regulators of cumulus cell function and oocyte quality. Human reproduction update. 2008;14(2):159-77. Russell DL, Robker RL. Molecular mechanisms of ovulation: co-ordination through the cumulus complex. Human reproduction update. 2007;13(3):289-312. Wathlet S, Adriaenssens T, Segers I, Verheyen G, Van de Velde H, Coucke W, et al. Cumulus cell gene expression predicts better cleavage-stage embryo or blastocyst development and pregnancy for ICSI patients. Human reproduction. 2011;26(5):1035-51. Gebhardt KM, Feil DK, Dunning KR, Lane M, Russell DL. Human cumulus cell gene expression as a biomarker of pregnancy outcome after single embryo transfer. Fertility and sterility. 2011;96(1):47-52 e2. Feuerstein P, Cadoret V, Dalbies-Tran R, Guerif F, Bidault R, Royere D. Gene expression in human cumulus cells: one approach to oocyte competence. Human reproduction. 2007;22(12):3069-77. Zhu X, Ye H, Fu Y. Use of Utrogestan during controlled ovarian hyperstimulation in normally ovulating women undergoing in vitro fertilization or intracytoplasmic sperm injection treatments in combination with a "freeze all" strategy: a randomized controlled dose-finding study of 100 mg versus 200 mg. Fertility and sterility. 2017;107(2):379-86 e4. Zhu X, Ye H, Fu Y. Duphaston and human menopausal gonadotropin protocol in normally ovulatory women undergoing controlled ovarian hyperstimulation during in vitro fertilization/intracytoplasmic sperm injection treatments in combination with embryo cryopreservation. Fertility and sterility. 2017;108(3):505-12 e2. Ferraretti AP, La Marca A, Fauser BC, Tarlatzis B, Nargund G, Gianaroli L. ESHRE consensus on the definition of 'poor response' to ovarian stimulation for in vitro fertilization: the Bologna criteria. Human reproduction. 2011;26(7):1616-24. The Istanbul consensus workshop on embryo assessment: proceedings of an expert meeting. Human reproduction. 2011;26(6):1270-83. Li CJ, Chen SN, Lin LT, Chern CU, Wang PH, Wen ZH, et al. Dehydroepiandrosterone Ameliorates Abnormal Mitochondrial Dynamics and Mitophagy of Cumulus Cells in Poor Ovarian Responders. J Clin Med. 2018;7(10). Zollner KP, Hofmann T, Zollner U. Good fertilization results associated with high IL-1beta concentrations in follicular fluid of IVF patients. The Journal of reproductive medicine. 2013;58(11-12):485-90. Rehman R, Jawed S, Zaidi SF, Baig M, Ahmeds K. Role of interleukin-l 3 in conception after intracytoplasmic sperm injection. JPMA The Journal of the Pakistan Medical Association. 2015;65(1):49-53. Kudo I, Murakami M. Prostaglandin E synthase, a terminal enzyme for prostaglandin E2 biosynthesis. Journal of biochemistry and molecular biology. 2005;38(6):633-8. Bayne RA, Eddie SL, Collins CS, Childs AJ, Jabbour HN, Anderson RA. Prostaglandin E2 as a regulator of germ cells during ovarian development. The Journal of clinical endocrinology and metabolism. 2009;94(10):4053-60. Nuttinck F, Marquant-Le Guienne B, Clement L, Reinaud P, Charpigny G, Grimard B. Expression of genes involved in prostaglandin E2 and progesterone production in bovine cumulus-oocyte complexes during in vitro maturation and fertilization. Reproduction. 2008;135(5):593-603. Takahashi T, Morrow JD, Wang H, Dey SK. Cyclooxygenase-2-derived prostaglandin E(2) directs oocyte maturation by differentially influencing multiple signaling pathways. The Journal of biological chemistry. 2006;281(48):37117-29. Niringiyumukiza JD, Cai H, Xiang W. Prostaglandin E2 involvement in mammalian female fertility: ovulation, fertilization, embryo development and early implantation. Reproductive biology and endocrinology : RB&E. 2018;16(1):43. Santos TA, El Shourbagy S, St John JC. Mitochondrial content reflects oocyte variability and fertilization outcome. Fertility and sterility. 2006;85(3):584-91. Tsai HD, Hsieh YY, Hsieh JN, Chang CC, Yang CY, Yang JG, et al. Mitochondria DNA deletion and copy numbers of cumulus cells associated with in vitro fertilization outcomes. The Journal of reproductive medicine. 2010;55(11-12):491-7. Lee SK, Zhao MH, Kwon JW, Li YH, Lin ZL, Jin YX, et al. The association of mitochondrial potential and copy number with pig oocyte maturation and developmental potential. The Journal of reproduction and development. 2014;60(2):128-35. Host E, Gabrielsen A, Lindenberg S, Smidt-Jensen S. Apoptosis in human cumulus cells in relation to zona pellucida thickness variation, maturation stage, and cleavage of the corresponding oocyte after intracytoplasmic sperm injection. Fertility and sterility. 2002;77(3):511-5. Corn CM, Hauser-Kronberger C, Moser M, Tews G, Ebner T. Predictive value of cumulus cell apoptosis with regard to blastocyst development of corresponding gametes. Fertility and sterility. 2005;84(3):627-33. Lee KS, Joo BS, Na YJ, Yoon MS, Choi OH, Kim WW. Cumulus cells apoptosis as an indicator to predict the quality of oocytes and the outcome of IVF-ET. Journal of assisted reproduction and genetics. 2001;18(9):490-8. Fang Y, Shang W, Wei DL, Zeng SM. Cited2 protein level in cumulus cells is a biomarker for human embryo quality and pregnancy outcome in one in vitro fertilization cycle. Fertility and sterility. 2016;105(5):1351-9 e4. Herrick JR, Brad AM, Krisher RL. Chemical manipulation of glucose metabolism in porcine oocytes: effects on nuclear and cytoplasmic maturation in vitro. Reproduction. 2006;131(2):289-98. Xie HL, Wang YB, Jiao GZ, Kong DL, Li Q, Li H, et al. Effects of glucose metabolism during in vitro maturation on cytoplasmic maturation of mouse oocytes. Sci Rep. 2016;6:20764. Spindler RE, Pukazhenthi BS, Wildt DE. Oocyte metabolism predicts the development of cat embryos to blastocyst in vitro. Molecular reproduction and development. 2000;56(2):163-71. Supplementary Files Additionalfile1SupplementalTableS1.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-12952","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research","associatedPublications":[],"authors":[{"id":324042,"identity":"49cea0e2-bb22-4990-a21a-f8517d5f0305","order_by":1,"name":"Li-Te Lin","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA6ElEQVRIiWNgGAWjYBACPmYg8cCAgYefvfngAyCbh4+QFjZmoJ4EAwYZyZ5jyQYgLWwEtTCAtDAw2BjcyDGTgIgQ0sLOf/BBQsEdHoMzB8wqv+bYyQANefjoBn6HMRskGDzjkTzekHZbdlsy0GFsxsY5+LWwSSQYHObhO3Pg2G3JbcxALTxs0gS0sP8AaWG4kdhWLLmtnigtbAwgLQI3ktkYP247TJQWY7DDgIHMLM247TgPGzMBv/DzH3z44cOfw/b87P0fP/7cVg1kND98jE8LCmDmAZPEKgcBxh+kqB4Fo2AUjIIRAwCWuUAyQ7DZWQAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0000-0001-5063-6793","institution":"Kaohsiung Veterans General Hospital","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Li-Te","middleName":"","lastName":"Lin","suffix":""},{"id":324043,"identity":"8f3479b3-ee05-416f-add2-d2fbb77d4e2a","order_by":2,"name":"Ju-Yueh Li","email":"","orcid":"","institution":"Kaohsiung Veterans General Hospital","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Ju-Yueh","middleName":"","lastName":"Li","suffix":""},{"id":324044,"identity":"ad0e60fe-22c9-44eb-9731-1ff2e167364e","order_by":3,"name":"Kuan-Hao Tsui","email":"","orcid":"","institution":"Kaohsiung Veterans General Hospital","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Kuan-Hao","middleName":"","lastName":"Tsui","suffix":""},{"id":324045,"identity":"903fd442-66e3-4f70-9924-d85310719233","order_by":4,"name":"Chia-Jung Li","email":"","orcid":"","institution":"Kaohsiung Veterans General Hospital","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Chia-Jung","middleName":"","lastName":"Li","suffix":""},{"id":324046,"identity":"01073dc1-0220-4d7c-acca-9a0fa27e8bb0","order_by":5,"name":"Peng-Hui Wang","email":"","orcid":"","institution":"Taipei Veterans General Hospital","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Peng-Hui","middleName":"","lastName":"Wang","suffix":""},{"id":324047,"identity":"2b4a11e5-1d90-451f-bd53-7388eb60c522","order_by":6,"name":"Zhi-Hong Wen","email":"","orcid":"","institution":"National Sun Yat-sen University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Zhi-Hong","middleName":"","lastName":"Wen","suffix":""}],"badges":[],"createdAt":"2020-01-30 12:07:30","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.2.22377/v1","doiUrl":"https://doi.org/10.21203/rs.2.22377/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":434894,"identity":"801e1c3c-3970-40f7-950d-cf707f71e298","added_by":"auto","created_at":"2020-01-31 16:59:58","extension":"tif","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":10124,"visible":true,"origin":"","legend":"mRNA levels of cumulus cell genes regarding inflammation (CXCL1, CXCL3, TNF, PTGES) between follicular phase ovarian stimulation group (Follicular) and luteal phase ovarian stimulation group (Luteal)","description":"","filename":"Fig1Inflammationrelatedgenes.tif","url":"https://assets-eu.researchsquare.com/files/56d51397-e9d6-4bd0-9ad9-6416605d8421/v1/Fig 1 Inflammation related genes.tif"},{"id":434895,"identity":"003f3087-9de1-47d3-8ca8-bc345b94ddf0","added_by":"auto","created_at":"2020-01-31 16:59:58","extension":"tif","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":13267,"visible":true,"origin":"","legend":"mRNA expression of cumulus cell genes regarding (a) oxidative phosphorylation (NDUFB7, NDUFA4L2, SLC25A27) and (b) apoptosis (DAPK3, BCL6B) between follicular phase ovarian stimulation group (Follicular) and luteal phase ovarian stimulation group (Luteal)","description":"","filename":"Fig2oxidationandapoptosisrelatedgenes.tif","url":"https://assets-eu.researchsquare.com/files/56d51397-e9d6-4bd0-9ad9-6416605d8421/v1/Fig 2 oxidation and apoptosis related genes.tif"},{"id":434896,"identity":"c5916698-ab42-4f06-9d05-f08c17d3d23d","added_by":"auto","created_at":"2020-01-31 16:59:59","extension":"tif","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":5980,"visible":true,"origin":"","legend":"mRNA levels of cumulus cell genes regarding glucose metabolism (PCK1, LDHC) between follicular phase ovarian stimulation group (Follicular) and luteal phase ovarian stimulation group (Luteal)","description":"","filename":"Fig3metabolism.tif","url":"https://assets-eu.researchsquare.com/files/56d51397-e9d6-4bd0-9ad9-6416605d8421/v1/Fig 3 metabolism.tif"},{"id":13487594,"identity":"1018ffeb-ff89-4cbd-856a-3c9377f12629","added_by":"auto","created_at":"2021-09-16 22:11:19","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":411784,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-12952/v1/e86bfea1-42d9-484f-bfcf-41d217ea3f0b.pdf"},{"id":434893,"identity":"b56b16b2-6395-4081-ab3f-f982ef6b18f8","added_by":"auto","created_at":"2020-01-31 16:59:58","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":20371,"visible":true,"origin":"","legend":"","description":"","filename":"Additionalfile1SupplementalTableS1.docx","url":"https://assets-eu.researchsquare.com/files/56d51397-e9d6-4bd0-9ad9-6416605d8421/v1/Additional file 1 Supplemental Table S1.docx"}],"financialInterests":"","formattedTitle":"Luteal phase ovarian stimulation versus follicular phase ovarian stimulation results in different human cumulus cells gene expression: a pilot study","fulltext":[{"header":"Introduction","content":" \u003cp\u003eLuteal phase ovarian stimulation (LPOS), referring to the initiation of ovarian stimulation from the luteal phase, has been regarded as a feasible protocol for in vitro fertilization (IVF) cycles (1) following the theory of multiple follicular recruitment waves in the same menstrual cycle had been proposed (2). LPOS was first applied in the fertility preservation of cancer patients (3, 4), and then used in the general infertile couples (5, 6). Studies showed that similar number of retrieved oocytes and mature oocytes and fertilization rate were noted between LPOS and follicular phase ovarian stimulation (FPOS) in the urgent fertility preservation (3, 4) or women with normal ovarian response (5, 6). In poor ovarian responders (PORs), some studies revealed that more competent oocytes and embryos could be obtained in the LPOS, compared to FPOS (7\u0026ndash;9). The possible rationale was that physiologic high levels of progesterone in the luteal phase could effectively block premature luteinizing hormone (LH) surge which more frequently occurred in the PORs during ovarian stimulation. Our previous study demonstrated that the numbers of retrieved oocytes, metaphase II oocytes, fertilized oocytes, and day-3 embryos were significantly higher in the LPOS group than in the FPOS group (7). However, some studies had conflicting results (10, 11). Furthermore, there was lack of large-scare randomized controlled trials to bolster the consequences. Therefore, no solid evidence support that PORs could really get more benefit from LPOS until now.\u003c/p\u003e \u003cp\u003eCumulus cells (CCs) are somatic cells surrounding the oocyte in cumulus-oocyte complexes (COCs). Bidirectional intercellular communication between CCs and the oocyte mediated by a network of specialized gap junctions is crucial for development of follicles (12). Oocyte-secreted factors, such as growth-differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15), generated from the oocyte regulate proliferation, apoptosis, luteinization, metabolism and expansion of CCs (13). CCs protect and nurture the oocyte, playing an essential role in oocyte maturation, ovulation and fertilization (14). Therefore, the expression profiles of CCs have the potential to reflect oocyte competence and even serve as predictors to determine embryo quality, pregnancy and live birth outcomes (15\u0026ndash;17).\u003c/p\u003e \u003cp\u003eAlthough progesterone can prevent premature luteinization effectively (18, 19), the influence of high levels of progesterone on oocytes or CCs were poorly understood. Therefore, the goal of this study was to investigate the differences of human CCs gene expression between LPOS and FPOS.\u003c/p\u003e "},{"header":"Materials And Methods","content":" \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eStudy Population and design\u003c/h2\u003e \u003cp\u003eThis prospective cohort study was implemented at the Reproductive Medicine Center of the Kaohsiung Veterans General Hospital from August 2017 to December 2018. We enrolled PORs undergoing IVF cycles. The inclusion criteria for POR in this study were defined according to the Bologna Criteria (20), having at least two of the three following features: (i) advanced maternal age (\u0026ge;\u0026thinsp;40\u0026nbsp;years) or any other risk factor for POR; (ii) a previous POR (\u0026le;\u0026thinsp;3 oocytes with a conventional stimulation protocol); (iii) an abnormal ovarian reserve test. An abnormal ovarian reserve test was defined as antral follicle count (AFC)\u0026thinsp;\u0026lt;\u0026thinsp;5 or anti-M\u0026uuml;llerian hormone (AMH)\u0026thinsp;\u0026lt;\u0026thinsp;1\u0026nbsp;ng/mL in this study. Furthermore, two episodes of a previous POR after maximal stimulation alone would be sufficient to define a patient as a POR. Patients were excluded if they had any of the following: (i) a diagnosis of primary ovarian insufficiency; (ii) a history of oophorectomy; (iii) a history of exposure to cytotoxic agents or pelvic irradiation for malignancy; (iv) a history of adjuvant supplementation or hormonal replacement therapy during the previous 3\u0026nbsp;months. The enrolled participants were then divided into two groups: follicular phase ovarian stimulation (FPOS) or luteal phase ovarian stimulation (LPOS). The choice of ovarian stimulation protocol was determined by physicians\u0026rsquo; consideration. Baseline and cycle characteristics and IVF outcomes were compared between the two groups.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eEthics Statement\u003c/h2\u003e \u003cp\u003eThis study was approved by the institutional review board of Kaohsiung Veterans General (VGHKS15-CT11-12) and Clinical Trial Register (ClinicalTrials.gov Identifier: NCT03238833). All participants were fully counselled and written informed consent was obtained. This study was performed adherence to approved guidelines and the Declaration of Helsinki.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eTreatment protocol\u003c/h2\u003e \u003cp\u003eIn FPOS group, ovarian stimulation with a 300\u0026nbsp;IU daily dose of combined recombinant follicle stimulating hormone (rFSH) plus recombinant LH (rLH) (Pergoveris, Merck Serono, Aubonne, Switzerland) was commenced within 5 days of the menstrual cycle. In LPOS group, spontaneous ovulation was confirmed by transvaginal sonography and progesterone level from day 15 to day 18 of the menstrual cycle. After confirmation of spontaneous ovulation, the women with at least one follicle of less than 8\u0026nbsp;mm started to undergo ovarian stimulation with a 300\u0026nbsp;IU daily dose of rFSH plus rLH (Pergoveris, Merck Serono, Aubonne, Switzerland).\u003c/p\u003e \u003cp\u003eIn both FPOS and LPOS groups, when the leading follicle reached 12\u0026nbsp;mm in diameter, the women received 0.25\u0026nbsp;mg of GnRH antagonist (Cetrotide; Merck Serono, Idron, France) daily until the day of oocyte trigger. Dual trigger, combination of recombinant human chorionic gonadotropin (rHCG) (Ovidrel, Merck Serono, Modugno, Italy) and GnRH agonist (Lupro, Nang Kuang Pharmaceutical Co., Ltd., Tainan, Taiwan), was administered when at least one dominant follicle reached the size of 17\u0026nbsp;mm. 36 hours after ovulation induction, oocyte retrieval was conducted by transvaginal ultrasound-guided needle aspiration.\u003c/p\u003e \u003cp\u003eOocytes were inseminated by intracytoplasmic sperm injection (ICSI) for all women in order to diminish the possibility of fertilization failure. Oocytes were denuded and inseminated if maturation status was verified by the presence of the first polar body. Fertilization was evaluated 18\u0026thinsp;~\u0026thinsp;20 hours after insemination and was defined success by the presence of two pronuclei. Embryos development and quality were assessed based on the number, symmetry of the blastomeres and embryonic fragmentation according to the criteria established by the Istanbul consensus workshop (21). All embryos were cryopreserved by vitrification on the third day after oocyte retrieval. An artificial frozen embryo transfer protocol was used for all participants. Oral estradiol (Ediol 8\u0026nbsp;mg, Synmosa Biopharma Corporation, Hsinchu County, Taiwan) was initiated on the third day of the menstrual cycle and endometrial thickness was monitored by transvaginal ultrasonography. When the endometrial thickness exceeded 8\u0026nbsp;mm, luteal support with progesterone intravaginal gel (Crinone 8% gel 90\u0026nbsp;mg/day, Merck Serono, Hertfordshire, UK) plus oral dydrogesterone (Duphaston 40\u0026nbsp;mg, Abbott, Olst, The Newtherlands) was added. Transabdominal ultrasound-guided embryo transplantation was performed 4 days after commencement of luteal support. The women underwent a pregnancy test 15 days after embryo transfer. If the pregnancy test was positive, progesterone was continued until 8\u0026thinsp;~\u0026thinsp;10 weeks of gestation. Clinical pregnancy was defined by the presence of fetal cardiac activity in an intrauterine gestational sac by transvaginal ultrasound. Live birth was determined by delivery of a live fetus after 20 weeks of gestation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eCumulus cells collection and genes expression\u003c/h2\u003e \u003cp\u003eCOCs were collected during oocyte aspiration and washed in the medium. CCs were removed mechanically using a sterile scalpel. CCs separated from the same patient\u0026rsquo;s COCs were pooled together for study. Isolated CCs were then transferred immediately into a sterile tube, centrifuged at 200\u0026nbsp;g for 5\u0026nbsp;min at room temperature and stored at -80\u0026nbsp;\u0026deg;C for further study.\u003c/p\u003e \u003cp\u003eCCs were analyzed for the expression of genes related to inflammation (CXCL1, CXCL3, TNF, PTGES), oxidative-phosphorylation (NDUFB7, NDUFA4L2, SLC25A27), apoptosis (DAPK3, BCL6B) and metabolism (PCK1, LDHC) using a real-time quantitative reverse-transcription polymerase chain reaction (qRT-PCR).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eRNA extraction and real-time qRT-PCR\u003c/h2\u003e \u003cp\u003eAs previously described (22), total RNA was extracted from CCs with the use of the TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer\u0026rsquo;s instructions. Each RNA pool was reverse transcribed to cDNA. To detect mRNA expression, real-time qRT-PCR analysis was performed using an ABI Prism 7700 Sequence Detection System (Perkin-Elmer Applied Biosystems, Foster City, CA, USA). PCR was performed using the SYBR Green PCR Core Reagents kit (Perkin-Elmer Applied Biosystems). Gene-specific qRT-PCR primers that were used are shown in supplemental Table S1. The thermal cycling conditions included an initial denaturation step at 95\u0026nbsp;\u0026deg;C for 10\u0026nbsp;min, 40 cycles at 95\u0026nbsp;\u0026deg;C for 15\u0026nbsp;s, and 60\u0026nbsp;\u0026deg;C for 1\u0026nbsp;min. Each set of qRT-PCR reactions was repeated three times. All of the samples with a coefficient of variation for Ct value\u0026thinsp;\u0026gt;\u0026thinsp;1% were retested. The GAPDH served as an internal control to normalize the expression of target genes. Relative expression levels were calculated for each sample after normalization against GAPDH.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eThe statistical analysis was carried out using the Statistical Package for Social Sciences (SPSS) version 22.0 (Chicago, IL, USA). The 2-tailed Student\u0026rsquo;s t-test were used to compare quantitative variables. The categorical variables were compared using Chi-Square tests or Fisher\u0026rsquo;s exact tests. The differences between groups were considered significant when the \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003ep\u003c/span\u003e value was less than 0.05.\u003c/p\u003e \u003c/div\u003e "},{"header":"Results","content":" \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eComparison of basic characteristics between FPOS and LPOS groups\u003c/h2\u003e \u003cp\u003eA total of 36 patients were recruited in this study and divided into FPOS (n\u0026thinsp;=\u0026thinsp;21) and LPOS (n\u0026thinsp;=\u0026thinsp;15) groups. The baseline characteristics in the two groups are presented in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. The mean age (39.7\u0026thinsp;\u0026plusmn;\u0026thinsp;3.8\u0026nbsp;years vs. 40.0\u0026thinsp;\u0026plusmn;\u0026thinsp;3.4\u0026nbsp;years) and body mass index (21.7\u0026thinsp;\u0026plusmn;\u0026thinsp;3.1\u0026nbsp;kg/m\u003csup\u003e2\u003c/sup\u003e vs. 23.6\u0026thinsp;\u0026plusmn;\u0026thinsp;3.8\u0026nbsp;kg/m\u003csup\u003e2\u003c/sup\u003e) of patients between the two groups were similar. Additionally, there were no statistically significant differences between groups in terms of infertility duration, previous IVF attempts, primary or secondary infertility, basal FSH, AFC and AMH.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cdiv class=\"SimplePara\"\u003e\u003cspan type=\"Bold\" class=\"Bold\" name=\"Emphasis\"\u003eBasic characteristics of poor ovarian responders undergoing follicular phase ovarian stimulation (FPOS) or luteal phase ovarian stimulation (LPOS)\u003c/span\u003e\u003c/div\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003eParameters\u003c/div\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003eFPOS\u003c/div\u003e \u003cdiv class=\"SimplePara\"\u003e(n\u0026thinsp;=\u0026thinsp;21)\u003c/div\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cdiv class=\"SimplePara\"\u003eLPOS\u003c/div\u003e \u003cdiv class=\"SimplePara\"\u003e(n\u0026thinsp;=\u0026thinsp;15)\u003c/div\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cdiv class=\"SimplePara\"\u003e\u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003ep\u003c/span\u003e value\u003c/div\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003eAge (years)\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003e39.7\u0026thinsp;\u0026plusmn;\u0026thinsp;3.8\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cdiv class=\"SimplePara\"\u003e40.0\u0026thinsp;\u0026plusmn;\u0026thinsp;3.4\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cdiv class=\"SimplePara\"\u003e0.818\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003eBody mass index (kg/m2)\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003e21.7\u0026thinsp;\u0026plusmn;\u0026thinsp;3.1\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cdiv class=\"SimplePara\"\u003e23.6\u0026thinsp;\u0026plusmn;\u0026thinsp;3.8\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cdiv class=\"SimplePara\"\u003e0.128\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003eInfertility duration (years)\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003e5.1\u0026thinsp;\u0026plusmn;\u0026thinsp;3.2\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cdiv class=\"SimplePara\"\u003e5.3\u0026thinsp;\u0026plusmn;\u0026thinsp;6.6\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cdiv class=\"SimplePara\"\u003e0.882\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003ePrevious IVF attempts (n)\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003e2.9\u0026thinsp;\u0026plusmn;\u0026thinsp;2.9\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cdiv class=\"SimplePara\"\u003e2.1\u0026thinsp;\u0026plusmn;\u0026thinsp;2.6\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cdiv class=\"SimplePara\"\u003e0.441\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003eTypes of infertility (%)\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cdiv class=\"SimplePara\"\u003e0.204\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003ePrimary infertility\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003e52.4\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cdiv class=\"SimplePara\"\u003e73.3\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003eSecondary infertility\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003e47.6\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cdiv class=\"SimplePara\"\u003e26.7\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003eBasal FSH (IU/l)\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003e5.6\u0026thinsp;\u0026plusmn;\u0026thinsp;4.0\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cdiv class=\"SimplePara\"\u003e5.4\u0026thinsp;\u0026plusmn;\u0026thinsp;2.2\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cdiv class=\"SimplePara\"\u003e0.864\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003eAntral follicle counts (n)\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003e4.4\u0026thinsp;\u0026plusmn;\u0026thinsp;1.4\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cdiv class=\"SimplePara\"\u003e4.9\u0026thinsp;\u0026plusmn;\u0026thinsp;1.7\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cdiv class=\"SimplePara\"\u003e0.273\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003eAnti-M\u0026uuml;llerian hormone (ng/ml)\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003e0.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cdiv class=\"SimplePara\"\u003e0.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.6\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cdiv class=\"SimplePara\"\u003e0.441\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"4\"\u003eData are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation or percentage.\u003c/td\u003e\u003c/tr\u003e \u003ctr\u003e\u003ctd colspan=\"4\"\u003eIVF, \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003ein vitro\u003c/span\u003e fertilization; FSH, follicle stimulation hormone\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eComparison of cycle characteristics and pregnancy outcome between FPOS and LPOS groups\u003c/h2\u003e \u003cp\u003eThe stimulation cycle outcomes of each group are shown in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. No statistically significant difference existed in duration of stimulation, total dose of gonadotrophins, or peak serum estradiol level. However, the peak serum progesterone level was significantly higher in LPOS group than FPOS group (6.8\u0026thinsp;\u0026plusmn;\u0026thinsp;6.8 vs. 0.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2, \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003ep\u003c/span\u003e\u0026thinsp;=\u0026thinsp;0.004).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cdiv class=\"SimplePara\"\u003e\u003cspan type=\"Bold\" class=\"Bold\" name=\"Emphasis\"\u003eCycle characteristics and pregnancy outcome of poor ovarian responders undergoing follicular phase ovarian stimulation (FPOS) or luteal phase ovarian stimulation (LPOS)\u003c/span\u003e\u003c/div\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003eParameters\u003c/div\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003eFPOS\u003c/div\u003e \u003cdiv class=\"SimplePara\"\u003e(n\u0026thinsp;=\u0026thinsp;21)\u003c/div\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cdiv class=\"SimplePara\"\u003eLPOS\u003c/div\u003e \u003cdiv class=\"SimplePara\"\u003e(n\u0026thinsp;=\u0026thinsp;15)\u003c/div\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cdiv class=\"SimplePara\"\u003e\u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003ep\u003c/span\u003e value\u003c/div\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003eStimulation duration (days)\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003e11.3\u0026thinsp;\u0026plusmn;\u0026thinsp;2.2\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cdiv class=\"SimplePara\"\u003e12.1\u0026thinsp;\u0026plusmn;\u0026thinsp;2.8\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cdiv class=\"SimplePara\"\u003e0.363\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003eGonadotropin dosage (IU)\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003e2882.1\u0026thinsp;\u0026plusmn;\u0026thinsp;690.1\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cdiv class=\"SimplePara\"\u003e2885.0\u0026thinsp;\u0026plusmn;\u0026thinsp;744.0\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cdiv class=\"SimplePara\"\u003e0.991\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003ePeak estradiol (pg/mL)\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003e749.9\u0026thinsp;\u0026plusmn;\u0026thinsp;553.2\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cdiv class=\"SimplePara\"\u003e756.7\u0026thinsp;\u0026plusmn;\u0026thinsp;671.5\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cdiv class=\"SimplePara\"\u003e0.975\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003eProgesterone (ng/ mL)\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003e0.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cdiv class=\"SimplePara\"\u003e6.8\u0026thinsp;\u0026plusmn;\u0026thinsp;6.8\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cdiv class=\"SimplePara\"\u003e0.004\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003eNo. of oocytes retrieved (n)\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003e3.0\u0026thinsp;\u0026plusmn;\u0026thinsp;1.4\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cdiv class=\"SimplePara\"\u003e3.1\u0026thinsp;\u0026plusmn;\u0026thinsp;1.6\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cdiv class=\"SimplePara\"\u003e0.713\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003eNo. of metaphase II oocytes (n)\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003e2.2\u0026thinsp;\u0026plusmn;\u0026thinsp;1.5\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cdiv class=\"SimplePara\"\u003e2.3\u0026thinsp;\u0026plusmn;\u0026thinsp;1.3\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cdiv class=\"SimplePara\"\u003e0.847\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003eMaturation rate (%)\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003e69.1\u0026thinsp;\u0026plusmn;\u0026thinsp;36.7\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cdiv class=\"SimplePara\"\u003e72.9\u0026thinsp;\u0026plusmn;\u0026thinsp;28.5\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cdiv class=\"SimplePara\"\u003e0.742\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003eNo. of fertilized oocytes (n)\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003e1.4\u0026thinsp;\u0026plusmn;\u0026thinsp;1.1\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cdiv class=\"SimplePara\"\u003e2.0\u0026thinsp;\u0026plusmn;\u0026thinsp;1.3\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cdiv class=\"SimplePara\"\u003e0.207\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003eFertilization rate (%)\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003e61.7\u0026thinsp;\u0026plusmn;\u0026thinsp;41.1\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cdiv class=\"SimplePara\"\u003e79.6\u0026thinsp;\u0026plusmn;\u0026thinsp;32.2\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cdiv class=\"SimplePara\"\u003e0.170\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003eNo. of Day 3 embryos (n)\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003e1.5\u0026thinsp;\u0026plusmn;\u0026thinsp;1.2\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cdiv class=\"SimplePara\"\u003e1.9\u0026thinsp;\u0026plusmn;\u0026thinsp;1.3\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cdiv class=\"SimplePara\"\u003e0.357\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003eNo. of top-quality Day 3 embryos (n)\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003e0.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.6\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cdiv class=\"SimplePara\"\u003e0.7\u0026thinsp;\u0026plusmn;\u0026thinsp;1.0\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cdiv class=\"SimplePara\"\u003e0.249\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003eClinical pregnancy rate (%)\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003e14.3\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cdiv class=\"SimplePara\"\u003e13.3\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cdiv class=\"SimplePara\"\u003e0.935\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003eLive birth rate (%)\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003e9.5\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cdiv class=\"SimplePara\"\u003e13.3\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cdiv class=\"SimplePara\"\u003e0.720\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"4\"\u003eData are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation or percentage.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eNo difference was observed between FPOS and LPOS groups regarding the number of retrieved oocytes (3.0\u0026thinsp;\u0026plusmn;\u0026thinsp;1.4 vs. 3.1\u0026thinsp;\u0026plusmn;\u0026thinsp;1.6, \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003ep\u003c/span\u003e\u0026thinsp;=\u0026thinsp;0.713), metaphase II oocytes (2.2\u0026thinsp;\u0026plusmn;\u0026thinsp;1.5 vs. 2.3\u0026thinsp;\u0026plusmn;\u0026thinsp;1.3, \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003ep\u003c/span\u003e\u0026thinsp;=\u0026thinsp;0.847), fertilized oocytes (1.4\u0026thinsp;\u0026plusmn;\u0026thinsp;1.1 vs. 2.0\u0026thinsp;\u0026plusmn;\u0026thinsp;1.3, \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003ep\u003c/span\u003e\u0026thinsp;=\u0026thinsp;0.207), embryos on Day 3 (1.5\u0026thinsp;\u0026plusmn;\u0026thinsp;1.2 vs. 1.9\u0026thinsp;\u0026plusmn;\u0026thinsp;1.3, \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003ep\u003c/span\u003e\u0026thinsp;=\u0026thinsp;0.357) and top-quality embryos on Day 3 (0.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.6 vs. 0.7\u0026thinsp;\u0026plusmn;\u0026thinsp;1.0, \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003ep\u003c/span\u003e\u0026thinsp;=\u0026thinsp;0.249). Moreover, the clinical pregnancy rate (14.3% vs. 13.3%, \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003ep\u003c/span\u003e\u0026thinsp;=\u0026thinsp;0.935) and live birth rate (9.5% vs. 13.3%, \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003ep\u003c/span\u003e\u0026thinsp;=\u0026thinsp;0.720) were similar between the two groups.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eCumulus cells gene expression between FPOS and LPOS groups\u003c/h2\u003e \u003cp\u003eAs shown in the Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, regarding inflammation related genes, CXCL1 (0.51 vs 1.00, \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003ep\u003c/span\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) and PTGES (0.30 vs 1.00, \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003ep\u003c/span\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01) mRNA expression was significantly lower in the LPOS group than in the FPOS group. However, mRNA expression of CXCL3 and TNF were not significantly different between the two groups.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn the Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, regarding genes related to oxidative-phosphorylation, NDUFB7 (0.12 vs 1.00, \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003ep\u003c/span\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) and NDUFA4L2 (0.33 vs 1.00, \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003ep\u003c/span\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01) were expressed at lower levels in the LPOS group than in the FPOS group. However, the mRNA expression of SLC25A27 was similar between the two groups. In terms of apoptosis related genes, DAPK3 (3.81 vs 1.00, \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003ep\u003c/span\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) and BCL6B (2.59 vs 1.00, \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003ep\u003c/span\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01) were more highly expressed in the LPOS group, compared to the FPOS group.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn terms of metabolism related genes (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e), compared to the FPOS group, significant increased PCK1 mRNA expression (3.13 vs 1.00, \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003ep\u003c/span\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) and decreased LDHC mRNA expression (0.12 vs 1.00, \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003ep\u003c/span\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) were found in the LPOS group.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e "},{"header":"Discussion","content":" \u003cp\u003eTo 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.\u003c/p\u003e \u003cp\u003eThis 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).\u003c/p\u003e \u003cp\u003eThe 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\u0026ndash;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\u0026ndash;35). Accordingly, this study seemed to reveal that LPOS may lead to mitochondrial dysfunction and increased apoptosis of CCs, causing adverse reproductive outcomes.\u003c/p\u003e \u003cp\u003eMoreover, 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\u0026ndash;39). Hence, LPOS seemed to harm oocyte competence and embryo growth by decreasing LDHC mRNA expression.\u003c/p\u003e \u003cp\u003eTaken 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\u0026ndash;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.\u003c/p\u003e \u003cp\u003eSeveral 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.\u003c/p\u003e \u003cp\u003eIn 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.\u003c/p\u003e "},{"header":"List of abbreviations","content":" \u003cp\u003eAFC: antral follicle count; AMH: anti-M\u0026uuml;llerian hormone; BCL6B: BCL6B transcription repressor; BMP15: bone morphogenetic protein 15; CC: cumulus cell; COC: cumulus\u0026ndash;oocyte\u0026ndash;complex; CXCL1: C-X-C motif chemokine ligand 1; DAPK3: death-associated protein kinase 3; FPOS: follicular phase ovarian stimulation; FSH: follicle stimulation hormone; GDF9: growth-differentiation factor 9; ICSI: intracytoplasmic sperm injection; IL-1: interleukin 1; IVF: in vitro fertilization; LDHC: lactate dehydrogenase C; LH: luteinizing hormone; LPOS: Luteal phase ovarian stimulation; NDUFA4L2: NDUFA4 mitochondrial complex associated like 2; NDUFB7:NADH:ubiquinone oxidoreductase subunit B7; PCK1: phosphoenolpyruvate carboxykinase 1; POR: poor ovarian responder; PTGES: prostaglandin E synthase; qRT-PCR: real-time quantitative reverse-transcription polymerase chain reaction; rFSH: recombinant follicle stimulating hormone; rHCG: recombinant human chorionic gonadotropin\u003c/p\u003e "},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe study conformed to the \u0026lsquo;\u0026lsquo;Declaration of Helsinki for Medical Research involving Human Subjects\u0026rsquo;\u0026rsquo;. Additionally, approval was obtained from the institutional review board at Kaohsiung Veterans General Hospital, with the identifier VGHKS15-CT11-12. The study was performed in accordance with approved guidelines.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding disclosure\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNo funding.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors' contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePH and ZH contributed conception and design of the study; LT and CJ organized the database and performed the statistical analysis; CJ did the laboratory research; LT and JY wrote the first draft of the manuscript; LT, JY and KH wrote sections of the manuscript. All authors contributed to manuscript revision, read and approved the submitted version.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was generously supported by grants VGHNSU107-013 from Kaohsiung Veterans General Hospital. We thank the Economy Company Ltd. for their technical and English editing service.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eKuang Y, Hong Q, Chen Q, Lyu Q, Ai A, Fu Y, et al. Luteal-phase ovarian stimulation is feasible for producing competent oocytes in women undergoing in vitro fertilization/intracytoplasmic sperm injection treatment, with optimal pregnancy outcomes in frozen-thawed embryo transfer cycles. Fertility and sterility. 2014;101(1):105-11.\u003c/li\u003e\n\u003cli\u003eBaerwald AR, Adams GP, Pierson RA. Ovarian antral folliculogenesis during the human menstrual cycle: a review. Human reproduction update. 2012;18(1):73-91.\u003c/li\u003e\n\u003cli\u003eCakmak H, Katz A, Cedars MI, Rosen MP. Effective method for emergency fertility preservation: random-start controlled ovarian stimulation. Fertility and sterility. 2013;100(6):1673-80.\u003c/li\u003e\n\u003cli\u003evon Wolff M, Thaler CJ, Frambach T, Zeeb C, Lawrenz B, Popovici RM, et al. Ovarian stimulation to cryopreserve fertilized oocytes in cancer patients can be started in the luteal phase. Fertility and sterility. 2009;92(4):1360-5.\u003c/li\u003e\n\u003cli\u003eMartinez F, Clua E, Devesa M, Rodriguez I, Arroyo G, Gonzalez C, et al. Comparison of starting ovarian stimulation on day 2 versus day 15 of the menstrual cycle in the same oocyte donor and pregnancy rates among the corresponding recipients of vitrified oocytes. Fertility and sterility. 2014;102(5):1307-11.\u003c/li\u003e\n\u003cli\u003eQin N, Chen Q, Hong Q, Cai R, Gao H, Wang Y, et al. Flexibility in starting ovarian stimulation at different phases of the menstrual cycle for treatment of infertile women with the use of in vitro fertilization or intracytoplasmic sperm injection. Fertility and sterility. 2016;106(2):334-41 e1.\u003c/li\u003e\n\u003cli\u003eLin LT, Vitale SG, Chen SN, Wen ZH, Tsai HW, Chern CU, et al. Luteal Phase Ovarian Stimulation May Improve Oocyte Retrieval and Oocyte Quality in Poor Ovarian Responders Undergoing In Vitro Fertilization: Preliminary Results from a Single-Center Prospective Pilot Study. Adv Ther. 2018;35(6):847-56.\u003c/li\u003e\n\u003cli\u003eWei LH, Ma WH, Tang N, Wei JH. Luteal-phase ovarian stimulation is a feasible method for poor ovarian responders undergoing in vitro fertilization/intracytoplasmic sperm injection-embryo transfer treatment compared to a GnRH antagonist protocol: A retrospective study. Taiwanese journal of obstetrics \u0026amp; gynecology. 2016;55(1):50-4.\u003c/li\u003e\n\u003cli\u003eLi Y, Yang W, Chen X, Li L, Zhang Q, Yang D. Comparison between follicular stimulation and luteal stimulation protocols with clomiphene and HMG in women with poor ovarian response. Gynecological endocrinology : the official journal of the International Society of Gynecological Endocrinology. 2016;32(1):74-7.\u003c/li\u003e\n\u003cli\u003eKansal Kalra S, Ratcliffe S, Gracia CR, Martino L, Coutifaris C, Barnhart KT. Randomized controlled pilot trial of luteal phase recombinant FSH stimulation in poor responders. Reproductive biomedicine online. 2008;17(6):745-50.\u003c/li\u003e\n\u003cli\u003eWu Y, Zhao FC, Sun Y, Liu PS. Luteal-phase protocol in poor ovarian response: a comparative study with an antagonist protocol. J Int Med Res. 2017:300060516669898.\u003c/li\u003e\n\u003cli\u003eZuccotti M, Merico V, Cecconi S, Redi CA, Garagna S. What does it take to make a developmentally competent mammalian egg? Human reproduction update. 2011;17(4):525-40.\u003c/li\u003e\n\u003cli\u003eGilchrist RB, Lane M, Thompson JG. Oocyte-secreted factors: regulators of cumulus cell function and oocyte quality. Human reproduction update. 2008;14(2):159-77.\u003c/li\u003e\n\u003cli\u003eRussell DL, Robker RL. Molecular mechanisms of ovulation: co-ordination through the cumulus complex. Human reproduction update. 2007;13(3):289-312.\u003c/li\u003e\n\u003cli\u003eWathlet S, Adriaenssens T, Segers I, Verheyen G, Van de Velde H, Coucke W, et al. Cumulus cell gene expression predicts better cleavage-stage embryo or blastocyst development and pregnancy for ICSI patients. Human reproduction. 2011;26(5):1035-51.\u003c/li\u003e\n\u003cli\u003eGebhardt KM, Feil DK, Dunning KR, Lane M, Russell DL. Human cumulus cell gene expression as a biomarker of pregnancy outcome after single embryo transfer. Fertility and sterility. 2011;96(1):47-52 e2.\u003c/li\u003e\n\u003cli\u003eFeuerstein P, Cadoret V, Dalbies-Tran R, Guerif F, Bidault R, Royere D. Gene expression in human cumulus cells: one approach to oocyte competence. Human reproduction. 2007;22(12):3069-77.\u003c/li\u003e\n\u003cli\u003eZhu X, Ye H, Fu Y. Use of Utrogestan during controlled ovarian hyperstimulation in normally ovulating women undergoing in vitro fertilization or intracytoplasmic sperm injection treatments in combination with a \"freeze all\" strategy: a randomized controlled dose-finding study of 100 mg versus 200 mg. Fertility and sterility. 2017;107(2):379-86 e4.\u003c/li\u003e\n\u003cli\u003eZhu X, Ye H, Fu Y. Duphaston and human menopausal gonadotropin protocol in normally ovulatory women undergoing controlled ovarian hyperstimulation during in vitro fertilization/intracytoplasmic sperm injection treatments in combination with embryo cryopreservation. Fertility and sterility. 2017;108(3):505-12 e2.\u003c/li\u003e\n\u003cli\u003eFerraretti AP, La Marca A, Fauser BC, Tarlatzis B, Nargund G, Gianaroli L. ESHRE consensus on the definition of 'poor response' to ovarian stimulation for in vitro fertilization: the Bologna criteria. Human reproduction. 2011;26(7):1616-24.\u003c/li\u003e\n\u003cli\u003eThe Istanbul consensus workshop on embryo assessment: proceedings of an expert meeting. Human reproduction. 2011;26(6):1270-83.\u003c/li\u003e\n\u003cli\u003eLi CJ, Chen SN, Lin LT, Chern CU, Wang PH, Wen ZH, et al. Dehydroepiandrosterone Ameliorates Abnormal Mitochondrial Dynamics and Mitophagy of Cumulus Cells in Poor Ovarian Responders. J Clin Med. 2018;7(10).\u003c/li\u003e\n\u003cli\u003eZollner KP, Hofmann T, Zollner U. Good fertilization results associated with high IL-1beta concentrations in follicular fluid of IVF patients. The Journal of reproductive medicine. 2013;58(11-12):485-90.\u003c/li\u003e\n\u003cli\u003eRehman R, Jawed S, Zaidi SF, Baig M, Ahmeds K. Role of interleukin-l 3 in conception after intracytoplasmic sperm injection. JPMA The Journal of the Pakistan Medical Association. 2015;65(1):49-53.\u003c/li\u003e\n\u003cli\u003eKudo I, Murakami M. Prostaglandin E synthase, a terminal enzyme for prostaglandin E2 biosynthesis. Journal of biochemistry and molecular biology. 2005;38(6):633-8.\u003c/li\u003e\n\u003cli\u003eBayne RA, Eddie SL, Collins CS, Childs AJ, Jabbour HN, Anderson RA. Prostaglandin E2 as a regulator of germ cells during ovarian development. The Journal of clinical endocrinology and metabolism. 2009;94(10):4053-60.\u003c/li\u003e\n\u003cli\u003eNuttinck F, Marquant-Le Guienne B, Clement L, Reinaud P, Charpigny G, Grimard B. Expression of genes involved in prostaglandin E2 and progesterone production in bovine cumulus-oocyte complexes during in vitro maturation and fertilization. Reproduction. 2008;135(5):593-603.\u003c/li\u003e\n\u003cli\u003eTakahashi T, Morrow JD, Wang H, Dey SK. Cyclooxygenase-2-derived prostaglandin E(2) directs oocyte maturation by differentially influencing multiple signaling pathways. The Journal of biological chemistry. 2006;281(48):37117-29.\u003c/li\u003e\n\u003cli\u003eNiringiyumukiza JD, Cai H, Xiang W. Prostaglandin E2 involvement in mammalian female fertility: ovulation, fertilization, embryo development and early implantation. Reproductive biology and endocrinology : RB\u0026amp;E. 2018;16(1):43.\u003c/li\u003e\n\u003cli\u003eSantos TA, El Shourbagy S, St John JC. Mitochondrial content reflects oocyte variability and fertilization outcome. Fertility and sterility. 2006;85(3):584-91.\u003c/li\u003e\n\u003cli\u003eTsai HD, Hsieh YY, Hsieh JN, Chang CC, Yang CY, Yang JG, et al. Mitochondria DNA deletion and copy numbers of cumulus cells associated with in vitro fertilization outcomes. The Journal of reproductive medicine. 2010;55(11-12):491-7.\u003c/li\u003e\n\u003cli\u003eLee SK, Zhao MH, Kwon JW, Li YH, Lin ZL, Jin YX, et al. The association of mitochondrial potential and copy number with pig oocyte maturation and developmental potential. The Journal of reproduction and development. 2014;60(2):128-35.\u003c/li\u003e\n\u003cli\u003eHost E, Gabrielsen A, Lindenberg S, Smidt-Jensen S. Apoptosis in human cumulus cells in relation to zona pellucida thickness variation, maturation stage, and cleavage of the corresponding oocyte after intracytoplasmic sperm injection. Fertility and sterility. 2002;77(3):511-5.\u003c/li\u003e\n\u003cli\u003eCorn CM, Hauser-Kronberger C, Moser M, Tews G, Ebner T. Predictive value of cumulus cell apoptosis with regard to blastocyst development of corresponding gametes. Fertility and sterility. 2005;84(3):627-33.\u003c/li\u003e\n\u003cli\u003eLee KS, Joo BS, Na YJ, Yoon MS, Choi OH, Kim WW. Cumulus cells apoptosis as an indicator to predict the quality of oocytes and the outcome of IVF-ET. Journal of assisted reproduction and genetics. 2001;18(9):490-8.\u003c/li\u003e\n\u003cli\u003eFang Y, Shang W, Wei DL, Zeng SM. Cited2 protein level in cumulus cells is a biomarker for human embryo quality and pregnancy outcome in one in vitro fertilization cycle. Fertility and sterility. 2016;105(5):1351-9 e4.\u003c/li\u003e\n\u003cli\u003eHerrick JR, Brad AM, Krisher RL. Chemical manipulation of glucose metabolism in porcine oocytes: effects on nuclear and cytoplasmic maturation in vitro. Reproduction. 2006;131(2):289-98.\u003c/li\u003e\n\u003cli\u003eXie HL, Wang YB, Jiao GZ, Kong DL, Li Q, Li H, et al. Effects of glucose metabolism during in vitro maturation on cytoplasmic maturation of mouse oocytes. Sci Rep. 2016;6:20764.\u003c/li\u003e\n\u003cli\u003eSpindler RE, Pukazhenthi BS, Wildt DE. Oocyte metabolism predicts the development of cat embryos to blastocyst in vitro. Molecular reproduction and development. 2000;56(2):163-71.\u003c/li\u003e\n\u003c/ol\u003e\n"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":" cumulus cells, follicular phase ovarian stimulation, gene expression, luteal phase ovarian stimulation, poor ovarian responders","lastPublishedDoi":"10.21203/rs.2.22377/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.2.22377/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"OBJECTIVE: Physiologic elevated levels of progesterone in luteal phase can impede early-onset LH surge. However, the impact of high levels of progesterone on the oocyte or cumulus cells (CCs) remains indistinct. Therefore, the aim of study was to investigate the CCs gene expression between luteal phase ovarian stimulation (LPOS) and follicular phase ovarian stimulation (FPOS) in poor ovarian responders (PORs) undergoing in vitro fertilization (IVF) cycles.\nMATERIALS AND METHODS: This was a prospective non-randomized trial (ClinicalTrials.gov Identifier: NCT03238833). A total of 36 PORs who conformed Bologna criteria and underwent IVF cycles were enrolled. 15 PORs were allocated to the LPOS group and 21 PORs were allocated to the FPOS group. Basic characteristics, cycle characteristics and pregnancy outcomes were compared between the two groups. Moreover, CCs genes regarding inflammation (CXCL1, CXCL3, TNF, PTGES), oxidative-phosphorylation (NDUFB7, NDUFA4L2, SLC25A27), apoptosis (DAPK3, BCL6B) and metabolism (PCK1, LDHC) were analyzed using real-time quantitative PCR between the two groups.\nRESULTS: Basic characteristics and IVF outcomes were similar between the two groups except significantly high progesterone level in the LPOS group. The mRNA expression of CXCL1 and PTGES were significantly lower in the LPOS group than in the FPOS group ( p \u003c 0.05). The LPOS group had significantly lower mRNA expression of NDUFB7 and NDUFA4L2 than the FPOS group ( p \u003c 0.05). DAPK3 and BCL6B mRNA expression were significantly higher in the LPOS group compared to FPOS group ( p \u003c 0.05). Increased expression of PCK1 and decreased expression of LDHC were observed in the LPOS group compared to the FPOS group. ( p \u003c 0.05).\nCONCLUSIONS: Compared to the FPOS, the LPOS seemed to reduce favorable inflammation and mitochondrial function, and induce apoptosis and abnormal glucose metabolism in CCs.","manuscriptTitle":"Luteal phase ovarian stimulation versus follicular phase ovarian stimulation results in different human cumulus cells gene expression: a pilot study","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2020-01-31 16:59:58","doi":"10.21203/rs.2.22377/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"e04796ef-5718-4241-bc5c-c1e87e1b7050","owner":[],"postedDate":"January 31st, 2020","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":54751,"name":"Cancer Biology"},{"id":54752,"name":"Sexual \u0026 Reproductive Medicine"}],"tags":[],"updatedAt":"2020-03-25T14:54:51+00:00","versionOfRecord":[],"versionCreatedAt":"2020-01-31 16:59:58","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-12952","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"identity":"rs-12952","version":["v1"]},"buildId":"k-LANOkFix9YAoV-Y-q_i","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}