Age-related decline in the expression of GDF9 and BMP15 genes in follicle fluid and granulosa cells derived from poor ovarian responders 

Age-related decline in the expression of GDF9 and BMP15 genes in follicle fluid and granulosa cells derived from poor ovarian responders  | 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 Help Center Sign In Submit a Preprint Cite Share Download PDF Research Age-related decline in the expression of GDF9 and BMP15 genes in follicle fluid and granulosa cells derived from poor ovarian responders Yan Gong, Jesse Li-Ling, Dongsheng Xiong, Jiajing Wei, Taiqing Zhong, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-29352/v3 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 04 Jan, 2021 Read the published version in Journal of Ovarian Research → Version 3 posted 4 You are reading this latest preprint version Show more versions Abstract Background: Growth differentiation factor 9 ( GDF9 ) and bone morphogenetic protein 15 ( BMP15 ) genes play important roles in folliculogenesis. Altered expression of the two have been found among patients with poor ovarian response (POR). In this prospective cohort study, we have determined the expression of the GDF9 and BMP15 genes in follicle fluid (FF) and granulosa cells (GCs) derived from poor ovarian responders grouped by age, and explored its correlation with the outcome of in vitro fertilization and embryo transfer (IVF-ET) treatment. Methods: A total of 196 patients with POR were enrolled from a tertiary teaching hospital. The patients were diagnosed by the Bologna criteria and sub-divided into group A ( 40 year old). A GnRH antagonist protocol was conducted for all patients, and FF and GCs were collected after oocyte retrieval. Expression of the GDF9 and BMP15 genes in the FF and GCs was determined with enzyme-linked immunosorbent assay (ELISA), quantitative real-time polymerase chain reaction (qRT-PCR) and Western blotting. Results: Compared with group C, groups A and B had significantly more two pronuclei (2PN) oocytes and transplantable embryos, in addition with higher rates of implantation and clinical pregnancy ( P < 0.05). The expression level of GDF9 and BMP15 genes in the FF and GCs differed significantly among the three groups ( P 0.05). The relative levels of GDF9 and BMP15 proteins in GCs have correlated with the relative mRNA levels in GCs and protein concentrations in FF ( P < 0.05). Conclusions: For poor ovarian responders, in particular those over 40, the expression of GDF9 and BMP15 is declined along with increased age and in accompany with poorer oocyte quality and IVF outcome, whilst the ratio of GDF9/BMP15 mRNA levels remained relatively constant. Sexual & Reproductive Medicine Cancer Biology GDF9 gene BMP15 gene Poor ovarian response in vitro fertilization Age Figures Figure 1 Introduction Poor ovarian response (POR) poses a great challenge for in vitro fertilization and embryo transfer (IVF-ET) treatment. Patients with POR have fewer retrieved oocytes , fewer transferable embryos, lower pregnancy rates, and greater odds for cycle cancellation and miscarriage [1] . With a prevalence of 9% ~ 24% in various IVF centers [2] , POR affects approximately 11.9% of Chinese women undergoing IVF -ET treatment [3] . Although various stimulation protocols and strategies have been proposed, patients with POR still bene fi t ed little from such treatment [4] . According to the Bologna criteria, POR is diagnosed with at least two of the following three criteria: advanced maternal age or any other risk factor for POR; previous history of POR; and an abnormal ovarian reserve test [5] . Although above criteria are useful for predicting ovarian response and counseling purpose, it has grouped together women with various etiologies and clinical characteristics [6] . Some have reported that, compared with elder patients, younger poor ovarian responders (< 40, especially < 35 year old ) have higher embryo quality and pregnancy rate but lower miscarriage rate [7-9] . A ge has therefore been proposed as an independent predictor for POR [7,8] . Studies have shown that elder poor responders, in particular those over 40, have significantly lower cumulative live birth rate (CLBR) compared with young poor responders (< 35) [10-12] . Therefore, 35 and 40 year s old have been us ed as the thresholds by the Poseidon Criteria and Bologna Criteria, respectively [5,6] . For elder poor responders , a ge-related infertility and decreased ovarian reserve (DOR) are the main obstacles, whereas for younger poor responders, ovarian ag ing seems to be independent of age. The heterogeneous prognosis may be attributable to various etiology of the patients. Risk factors for POR include fecundity decline with age, ovarian cystectomy, chronic smoking, genetic factors, previous chemotherapy and/or radiotherapy , etc. [13] . The pathophysiology of POR is complex and may involve declined endocrine feedback of ovarian factors [14-16] . Oocyte-secreted factors (OSFs) can regulate follicle development [17] . The transforming growth factor β (TGFβ) superfamily is one of the most important OSFs which control the function of granulosa cells (GCs) . The latter in turn can nourish the oocyte [18] . Growth differentiation factor 9 ( GDF9 ) and bone morphogenetic protein 15 ( BMP15 , also known as GDF9 B ) are essential members of the TGF-β superfamily which can enhance the proliferation and metabolism of the GCs and stimulat e the expression of kit ligand (KL) in GCs. The GCs can then provide nutrients, steroid hormones and growth factors which can enable oocyte maturation via an intimate cross-talk [15,17] . Cumulus expansion is a compl ex process which is critical for oocyte maturation and ovulation [19] . The process is highly dependent on the interactions of two signals released by epidermal growth factor (EGF)-like peptides and OSFs [20] . Previous studies have shown that genetic variants and abnormal expression of GDF9 and BMP15 may predispose to follicle atresia and early exhaustion of ovarian reserve [15,16] . Altered expression of the two have been found in patients with premature ovarian insufficiency (POI), DOR and infertility [14-16,21] . Moreover, for patients undergoing IVF-ET treatment, GDF9 and BMP15 in follicular fluid (FF) and GCs have been associated with poorer oocyte quality and outcome of treatment [22] , hence were proposed as biomarkers for the potential of oocyte development [22,23] . Considering the important roles of GDF9 and BMP15 in folliculogenesis , this prospective study was designed to analyze the expression of GDF9 and BMP15 in the FF and GCs from patients with POR from more homogeneous age groups . Identification of the underlying mechanism may facilitate design of individualized treatment protocols for subgroups of patients with POR. Methods Population This study was registered with the Chinese Clinical Trial Registry Center (Registration No. ChiCTR1800016107) a nd approv ed by the Medical Ethics Committee of Sichuan Provincial Women ’ s and Children ’ s Hospital . All patients had given written informed consent. The study also conformed to the Declaration of Helsinki for Medical Research involving Human Subjects (2013 revision). Ethnic Han Chinese patients diagnosed with POR by the Bologna criteria [1] were enrolled from May 2018 to October 2019 and divided into three groups: group A ( 40 year old). All patients underwent IVF-ET treatment. Patients were excluded from the study should they me e t any of the following criteria: (1) C ongenital uterine malformation, endometriosis, polycystic ovarian syndrome, intrauterine adhesion, single ovary; (2) S ystemic lupus erythematosus and/or sicca syndrome; (3) U ncontrolled endocrinopathy such as diabetes, hyper / hypothyroidism, and hyperprolactinemia; (4) A bnormal chromosomal karyotype ; (5) C ontrolled ovarian stimulation (COS) in the past three months; and (6) I ntracytoplasmic sperm injection (ICSI) cycle due to male factor infertility. Medical his tory w as taken for all participants, including regularity of menstrual cycle, duration of infertility and pre-treatment protocols. H eight and body weight (with shoes and heavy clothing taken off ) were measured. Body weight index (BMI) was calculated as weight divided by height squared (kg/m 2 ). COS and IVF procedures A GnRH antagonist protocol was conducted for all patients. F rom day 2 or 3 of the cycle, 187.5 ~ 225 IU/d human recombinant follicle stimulating hormone ( rFSH ) ( Gonal -F, Merck Serono, Germany; Puregon ®, Merck Sharp & Dohme, USA) and 75 IU/d human menopausal gonadotrop h in ( hMG ) ( menotrophin , Livzon Pharmaceutical Group Inc, Zhuhai, China ) were injected. The dosage of rFSH and hMG was adjusted according to the ovarian response. Ganirelix ( Merck Sharp & Dohme, USA ) was administered should one of the following criteria be met: serum estradiol (E 2 ) > 300 pg /ml, follicle diameter > 14 mm, luteinizing hormone (LH) > 10 IU/L . When the leading follicle reached 18 mm in diameter, 10 000 IU of urinary human chorionic gonadotrophin ( u h CG ) ( Livzon Pharmaceutical Group Inc, Zhuhai, China) was injected to trigger the ovulation. Oocytes were retrieved by trans-vaginal ultrasound guidance within approximately 36 h after the trigger, and follicle flushing was not used . Oocytes were fertilized by conventional IVF for 4 ~ 6 h . Mature oocyte was defined as being at the metaphase Ⅱ ( MⅡ ) stage with the first polar body visible in the cytoplasm. 17 ~ 18 h after the IVF, normal fertilized oocyte was confirmed should it contain two pronuclei (2PN). Cultured embryos were evaluated on day 3 based on the number of blastomeres and degree of fragmentation . Embryos of grade A ~ C on day 3 were defined as transplantable embryos [24] . One or two transplantable embryos were transferred. Luteal phase support was started on the oocyte retrieval day with the injection of 60 mg/d progesterone oil (Zhejiang Xianju Pharmaceutical Co., Ltd. Taizhou, China) or vaginal progesterone ( Crinone 8% gel, Merck, Germany). The reasons for c ancel ed cycle s included follicular growth failure (10 days after COS, leading follicle diameter 2.5 ng/ml on the trigger day. C linical pregnancy was defined as detection of embryonic heartbeat. The r ates of implantation, clinical pregnancy, multiple pregnancy, and miscarriage were calculated [8] . Measurement of basal endocrine parameters in serum E ndocrine parameters including E 2 , progesterone (P), total testosterone (TT), prolactin (PRL), FSH and LH were measured with an electrochemiluminescence immunoassay platform (Roche Diagnostics GmbH, Mannheim, Germany) . Anti-M ü llerian hormone (AMH) was measured with an enzyme - linked immunosorbent assay kit (Guangzhou Kangrun Biotech, Co., Ltd, Guangdong, China). I ntra - and inter-assay coefficients for the above varia ble s were set as < 5% and 10%, respectively. Collection of FF and GCs FF samples were carefully collected from follicles with a diameter of ≥ 18 mm, and centrifug ed immediately at 700 × g for 5 min . T he supernatant was stored at -80 ℃. GCs were obtained by follicular aspiration and isolated from blood cells and cellular debris with a lymphocyte separation medium ( Beijing Solarbio Science and Technology Corporation, Beijing, China) by centrifugation at 700 × g for 10 min. R esidual red blood cells were removed with a red blood cell lysis buffer ( Solarbio Science and Technology Corporation , Beijing, China ). The GCs were stored at - 80℃ until the time of use. For each patient, the FF and GCs were collected from all follicles and pooled as one sample. Determination of GDF9 and BMP15 in FF by ELISA The concentrations of GDF9 and BMP15 in FF were measured with a commercial enzyme-linked immunosorbent assay kit ( Elabscience Biotechnology Co., Ltd., Wuhan, China) by following the manufacturer’s instructions. The standard product in GDF9 assay kit was human GDF9 recombinant protein expressed in E. coli , and the sequence was Gly320-Arg 454. The kit has been crossed experimented with human GDF-2, GDF-7, GDF-10, GDF-11, GDF-15 and BMP-15 , with t he crossov er rates being 0.74%, 0.61%, 0.78%, 0.65%, 0.72% and 0.41%, respectively. The standard product of the BMP15 assay kit wa s human BMP15 recombinant protein expressed in E. coli , and the sequence was Gln268-Arg392. The kit has been crossed experimented with human BMP-1, BMP-2, BMP-4, BMP-6, BMP-7 and GDF9 , with the crossover rates being 0.53%, 0.92%, 0.79%, 0.56%, 0.74% and 0.38%, respectively. The standard use of the two kits was to make serial dilutions, i.e., 1000, 500, 250, 125, 62.5, 31.25, 15.63, 0 pg /ml, of a working solution of 1000 pg /ml before the measurement. The FF samples were thawed and mixed together, and 100 μ l FF was added to each well in duplicate. The absorbance value was measured at 450 nm with a Perlong DNM-9602G microplate spectrophotometer ( Perlong New Technology Co., Ltd., Beijing, China). The sensitivity of the assay was set as 9.38 pg /ml. The detection range of GDF9 and BMP15 w as set as 15.36 ~ 1000 pg /ml. The concentration of GDF in some FF samples were above the range. Such samples were diluted 2 times, and 100 μ l diluted FF was then added to each well. The final concentration was calculated by multipl ying the detection value with the dilution factor. Determination of mRNA expression in GCs by quantitative real-time polymerase chain reaction ( qRT -PCR) Total RNA was isolated from GCs with a RNAprep Pure Micro Kit ( Tiangen Biotech Co., Ltd., Beijing, China). The purity and concentration of RNA were determined with Nanodrop-2000 (Thermo Fisher Scientific, Waltham, Massachusetts, USA) under the absorbance of 260 nm/280 nm. The RNA was reversely transcribed into cDNA using a PrimeScript TM RT Reagent Kit with gDNA Eraser ( TaKaRa , Tokyo, Japan). The cDNA was then amplified using TB Green TM Premix Ex Taq TM Ⅱ ( TaKaRa , Tokyo, Japan) by qRT -PCR in triplicate. The PCR conditions were 95℃ for 30 s, followed by 40 cycles of 95℃ for 10 s, 60℃ for 30 s, and 65℃ for 5 s. Formation of a single product was verified with a melting curve method. GAPDH , β-ac tin and HPRT were used as internal controls . The mRNA levels of the target genes were calculated with the 2 -ΔCT method and expressed as fold change relative to the geometric means of the three internal controls. All PCR reactions were conducted in triplicate . Primers used in the qRT -PCR are shown in Table 1. Table 1 Sequences of primers used in qRT -PCR Gene Primer (5'→3') Annealing temperature (℃) Efficienc y GDF9 F: TGGAGCATCCTTCAGCAC R: GCAGCCTCTTCTCCCACA 57.2 9 8.8 % BMP15 F: TTTACCGCCATCATCTCCAA R: TTTCCAAGCGTTAGACATCA 53.4 9 1.9 % GAPDH F: ACGGATTTGGTCGTATTGGG R: CGCTCCTGGAAGATGGTGAT 57.4 1 01.6 % β -actin F: GACAGGATGCAGAAGGAGAT R: CTGCTTGCTGATCCACATCT 5 3.5 99.6% H PRT F: CCATTCCTATGACTGTAGAT R: CCAGTTAAAGTTGAGAGATCAT 5 1.8 96.5% Determination of GDF9 and BMP15 in GCs by Western blotting GCs were lysed in RIPA lysis buffer ( KeyGen Biotech Co., Ltd., Nanjing, China) containing the Halt TM Protease Inhibitor Cocktail (Invitrogen, Karlsruhe, Germany). A BCA protein quantitative kit (Pierce, Thermo Fisher Scientific, Rockford, IL, USA) was used for determin ing the protein concentration. The total proteins (60 µg/lane) were subjected to 10% SDS-PAGE and transferred onto a polyvinylidene fluoride (PVDF) membrane (EMD Millipore, Billerica, MA, USA). The membrane was then blocked in TBST (Tris-buffered saline with Tween-20) containing 5% BSA (Bio-Rad, Hercules, CA, USA) at room temperature for 1 h, and incubated with primary antibody overnight at 4 ℃ . The p rimary antibodies used in this study have include d GDF9 (1:500, Beyotime Biotechnology Co., Ltd., Shanghai, China), BMP15 (1:500, Beyotime Biotechnology Co., Ltd.), and GAPDH (1:2000, Bioss , Beijing, China). After washed with TBST for three times, the membranes were incubated with secondary antibodies for 2 h at room temperature. The protein was then detected with a SuperSignal ® West Pico Trial Kit (Thermo Scientific Pierce, IL, USA). Band intensity was measured with a Gel Doc XR densitometer (Bio-Rad, Hercules, CA, USA) and normalized with that of the internal control. Statistical analysis Data were analyzed with SPSS 17.0 software (SPSS Inc., Chicago IL, USA). Continuous variables were expressed as mean ± standard deviation (SD). The Kolmogorov-Smirnov test was used to assess the normality of data distribution. Continuous variables with normal distribution were compared using one-way ANOVA with post hoc Bonferroni test . Categorical data were compared using Chi-squared test. Pearson’s correlation was used to assess the correlation between the relative mRNA levels in GCs and protein concentration s in FF. Significance level was set as < 0.05, and two-tailed test was used for all hypothesis tests. Linear regression equation was established for the expression levels of GDF9 against BMP15 mRNA in each sample. Results Baseline characteristics of the patients Baseline characteristics of the patient are shown in Table 2. The average age s of the three groups have differed significantly ( P < 0.05). Compared with group A, the duration of infertility was significantly longer in group C ( P 0.05). Table 2 Baseline characteristics of the patients group A (n = 63) Group B (n = 65) Group C (n = 68) Age (years) abc 30.65 ± 2.57 37.37 ± 1.53 42.59 ± 1.70 Abnormal menstrual cycle 34.92% (22/63) 36.92% (24/65) 35.29% (24/68) Duration of infertility ( year s) b 3.32 ± 2.21 4.03 ± 3.05 4.68 ± 3.00 BMI (kg/m 2 ) 22.08 ± 2.62 22.21 ± 2.00 22.44 ± 2.62 AFC 4.05 ± 1.43 3.91 ± 1.69 3.94 ± 1.68 FSH/LH ratio 2.68 ± 1.29 2.76 ± 1.71 2.99 ± 1.21 E 2 ( pg /ml ) 76.12 ± 34.71 77.49 ± 52.67 85.08 ± 50.88 P ( ng/ml ) 0.77 ± 0 .35 0.78 ± 0 .33 0.84 ± 0 .52 TT ( ng/ml ) 0.37 ± 0 .15 0.36 ± 0 .15 0.39 ± 0 .10 AMH ( ng/ml ) 0.80 ± 0.39 0.78 ± 0.24 0.73 ± 0.39 PRL ( ng/ml ) 199.84 ± 39.35 205.58 ± 58.49 197.26 ± 59.45 Data are presented as mean ± SD or percentage (number). Abbreviations: BMI Body mass index, AFC Antral follicle count, FSH Follicle stimulating hormone, LH Luteinizing hormone, E 2 Estradiol, P Progesterone, TT Total testosterone, AMH Anti-M ü llerian hormone, PRL Prolactin. The rate of abnormal menstrual cycle was compared with Chi-square test. P < 0.05 was considered as statistically significant. a P < 0.05, group A vs. group B b P < 0.05, group A vs. group C c P < 0.05, group B vs. group C Decline of oocyte quality and IVF outcome with increased age In total t hirty-one cycles were canceled. The main reasons included follicular growth failure (10 cycles), failed oocyte retrieval at the time of follicle aspiration (6 cycles), no transplantable embryos (5 cycles ), a c cumulation of embryos (5 cycles), and progesterone > 2.5 ng/ml on the trigger day (5 cycles). Compared with group C, groups A and B had significantly more 2PN oocytes, transplantable embryos, and higher rates of implantation and clinical pregnancy ( P < 0.05). The dosage of rFSH , duration of COS, endometrial thickness and E 2 levels on the trigger day, the number of retrieved oocytes, MⅡ oocytes and embryos per ET, and the rates of miscarriage and multiple pregnancy did not significantly differ among the three groups ( P > 0.05) (Table 3). Table 3 COS, IVF outcomes and biochemical markers in FF group A (n = 63) Group B (n = 65) Group C (n = 68) COS Dosage of rFSH (IU) 2476.31 ± 379.34 2556.69 ± 419.57 2586.90 ± 587.63 Duration of COS (d) 9.89 ± 2.85 9.68 ± 2.22 9.33 ± 2.34 E 2 on trigger day ( pg /mL) 756.95 ± 191.65 740.23 ± 180.47 721.03 ± 151.62 Endometrial thickness (mm) 9. 10 ± 1.89 8.95 ± 1.91 8.81 ± 1.61 N umber of oocytes retrieved 2.98 ± 0.67 2.82 ± 0.87 2.68 ± 0.90 N umber of MⅡ 2.54 ± 0.94 2.43 ± 0.79 2.37 ± 0.74 N umber of 2PN ab 2.08 ± 0.93 1.87 ± 0.87 1.36 ± 0.58 N umber of transplantable embryos ab 2.02 ± 0.57 1.85 ± 0.71 1.22 ± 0.49 Cancel cycle rate (%) 11.11 % (7/63) 12.31% (8/65) 23.53% (16/68) ET N umber of e mbryos per ET 1.43 ± 0.50 1.44 ± 0.50 1.33 ± 0.47 Implantation rate (%) ab 30.00% (24/80) 24.39% (20/82) 10.14% (7/69) Clinical pregnancy rate (%) ab 37.50% (21/56) 29.82% (17/57) 11.54% (6/52) Miscarriage rate (%) 9.52% (2/21) 23.53% (4/17) 33.33% (2/6) Multiple pregnancy rate (%) 14.29% (3/21) 17.65% (3/17) 16.67% (1/6) In FF GDF9 ( pg /ml) abc 1020 ± 117 904 ± 70 671 ± 53 BMP15 ( pg /ml) abc 666 ± 40 643 ± 46 420 ± 40 Data are presented as mean ± SD or percentage (number). Abbreviations: COS Controlled ovarian stimulation, E 2 Estradiol, ET Embryos transferred, FF Follicle fluid, GDF9 Growth differentiation factor 9, BMP15 Bone morphogenetic protein-15. Chi-squared test was used to compare the rates of maturation , fertilization, cleaved embryo, higher quality embryo, cancel cycle, implantation, clinical pregnancy, miscarriage and multiple pregnancy between the two groups. P < 0.05 was considered as statistically significant. a P < 0.05, group A vs. group C b P < 0.05, group B vs. group C c P < 0.05, group A vs. group B Decreased expression of GDF9 and BMP15 in FF with increased age As shown in Table 3, the concentration of GDF9 ( 101 2 ± 117 p g/ml vs. 90 4 ± 70 p g/ml vs. 671 ± 5 3 p g/ml ) and BMP15 ( 666 ± 40 p g/ml vs. 64 3 ± 4 6 p g/ml vs. 4 20 ± 40 p g/ml ) in FF have differed significantly between the three groups ( P < 0.05), with an obvious trend of decline with increased age. Decreased expression of GDF9 and BMP15 in GCs with increased age Only GCs in FF with oocyte were detected for mRNA and protein. Therefore, there were 61, 60 and 59 samples from group A, group B and group C, respectively. The CT values of housekeeping genes did not differ significantly among the three groups ( P > 0.05). As shown in Figures 1A and 1 B, the expression of GDF9 and BMP15 in GCs , at the levels of both mRNA and protein, have differed significantly between the three groups ( P < 0.05) . Both showed a trend of decline with increased age. As shown in Figure 1C, the single band at 51 kDa represented GDF9 , while the single band at 45 kDa represented BMP15 . As shown in Table 4, t he relative protein levels in GCs w as positively correlated with the relative mRNA levels in GCs and protein concentration s in FF by Pearson’s correlation analysis ( P < 0.05). Table 4 Correlation between the proteins and mRNA levels relative protein levels in GCs relative mRNA levels in GCs protein concentration in FF r P r P GDF9 0.704 < 0.001 0.501 0.001 BMP15 0.737 < 0.001 0.701 < 0.001 The ratio of GDF9/BMP15 mRNA levels The ratio of GDF9/BMP15 mRNA levels of the three groups were compared using one-way ANOVA. The ratio of GDF9/BMP15 mRNA levels for groups A, B and C were 1.23 (1.00-1.35), 1.23 (1.08-1.38), and 1.20 (1.09-1.33), respectively. N o significant difference was detected between the groups ( P > 0.05). Linear regression equation for the mRNA levels of GDF9 against BMP15 in e ach sample was established as y = 1.097 x + 0.047. Discussion We have proven t hrough this study that the oocyte quality and IVF outcome will decrease with increased age among patients with POR, in particular those over 40, which also correlated with decline of GDF9 and BMP15 expression in FF and GCs. The ratio of GDF9/BMP15 mRNA levels has remained to be relatively constant, and the protein and mRNA levels were positively correlated in both FF and GCs. Although the quantity of oocyte was poor in all groups in this study, the numbers of normal fertilized oocytes and transplantable embryos have decreased steeply to 1.36 and 1.22 in poor responders over 40 , respectively . And the oocyte quality is one of the most critical factors for pregnancy. Similar to previous reports, the rates of implantation and clinical pregnancy in elder patients (> 40) have decreased to 10.14% and 11.54%, respectively [7,8] . This seems to have reflected the poor quantity and quality of the oocytes from elder poor responders whom may benefit less from IVF treatment. For poor responders between 35 and 40, the rates of implantation and clinical pregnancy were 24.39% and 29.82%, respectively . B oth were lower than those of under 35 , albeit with no statistical significance. Researchers have p revious ly proposed that 35 - 37 year of age to be the threshold for decreased rate of euploidy embryos and live births [25] . Such patients should therefore consider IVF treatment earlier. Meanwhile, younger poor responders (< 35) still have reasonable number of transplantable embryos (2.02 ± 0.57) and pregnancy rate (37.50%), and should be actively treated. Of note, the miscarriage rate may also rise with increased age [8] . The underlying mechanisms for age-related decline of oocyte quality and IVF outcome are variable, including chromosomal aberration in oocytes and embryos due to advanced maternal age [26-28] . The autocrine function of oocytes in POR is also important but not yet fully understood. GDF9 and BMP15 secreted by the oocyte play an important role in the cross-talk between cumulus GCs and the oocytes [22] . The functions of GDF9 and BMP15 in follicular development include suppressing GCs apoptosis and promoting cell proliferation [29] , enhancing the effect of FSH and insulin-like growth factor- I (IGF- I) on GCs, then providing more E 2 and glycolysis for oocyte [30,31] . GDF9 and BMP15 can also prevent premature luteinization and promote normal expansion of cumulus cells till the LH surge [32,33] . In turn, GCs are necessary to nourish the oocytes and promote their maturation [17,34] . Therefore, altered expression and/or function of GDF9 and BMP15 may result in abnormal folliculogenesis and poor oocyte quality. Declined expression of the GDF9 and BMP15 genes has previously been discovered among patients with DOR [21,35] . In this study, we further proved that such de cline is associated with increased age, particularly for those over 40. FF and GCs form the microenvironment of the oocytes, and certain components of them may reflect the metabolism and endocrine status of the oocyte [34,36] . In this study, we found that the relative protein levels in GCs have correlate d with the relative mRNA levels in GCs and protein concentration in FF. This has reflected the si milar changes of the two proteins in FF and GCs , both of which can reflect the status of oocyte. Furthermore, the decline of GDF9 and BMP15 expression was accompanied with poor oocyte quality and IVF outcome in elder patients. Compared with group A (< 35), group B (35 - 40) had significantly lower expression. The ir oocyte quality and IVF outcome were also poorer albeit with no statistical significance. Hence, GDF9 and BMP15 may provide more sensitive and earlier biomarkers for the fertility of poor ovarian responders. Declined expression of the two may, at least in part, account for the age-related poor oocyte quality in poor responders. Physiologically, GDF9 and BMP15 can promote the expansion of cumulus GCs till the LH surge [32,33] . For elder poor responders, the steep dec line in GDF9 and BMP15 expression may affect the response of GCs to LH surge. For such patients, exogenous LH in conjunct with rFSH may compensate the insufficient function of LH and improve the outcome of IVF-ET treatment [37] . Of note , the ratio of GDF9/BMP15 mRNA levels in GCs has remained relatively constant among the three groups , and t herefore m ay not be associ ate d with the onset of early DOR. Crawford et al [38] also found the ratio of GDF9 / BMP15 mRNA to be strongly reserved in each species but differed markedly between species. Oocyte derived from poly-ovulatory species exhibited higher GDF9 / BMP15 ratios, e.g., mouse (5.19) and rat (3.65), compared with species with low ovulation rates, e.g., sheep (1.26), cow (0.24) and deer (0.10) [38] . In the present study, t he ratio of human GCs (1.20-1.23) was similar to that of low o vulatory species such as sheep (1.26). GDF9 and BMP15 work synergistic ally to influence the proliferation of GCs, and GCs of each species might have evolved to respond to unique GDF9 / BMP15 ratios [39] . Nevertheless , Crawford et al. found the ratio in pig oocyte s (poly-ovulatory species) to be also low (0.51) [38] . Therefore, t he ratio of GDF9 / BMP15 may not be the only factor which can influence the ovulation rate. The underlying mechanism of the GDF9 / BMP15 ratio and ovulation rate demands further research. The limitation of this study lies in its relatively small sample size and lack of livebirth rate d ue to the limited study period. Moreover, as the main purpose of this study was to evaluate GDF9 and BMP15 expression in female age groups, we have excluded ICSI cycles due to male factors. The latter, in particular advanced paternal age, may also affect the embryo quality and outcome of IVF -ET treatment. Conclusions In summary, we have discovered an age-related decline in GDF9 and BMP15 expression among patients with POR, which may in part account for the age-related poor oocyte quality and IVF outcome. Identification of the heterogeneity may facilitate design of individualized protocols to attain better treatment outcome and avoid repeated cycles. L arger cohort studies are required to validate the result s of this study. Researchers have recently suggested that exogenous recombinant GDF9 and/or BMP15 can enhance blastocyst formation during IVF treatment [40,41] . Should such strategy be adapted for poor ovarian responders, dosage s dependent on age and basal levels of GDF9 and BMP15 expression may be considered. Declarations Acknowledgement We thank our colleagues at the Reproductive Medicine Center of Sichuan Provincial Women’s and Children’s Hospital for their assistance in sample collection. We thank Xiang y u Li and ShiQi Chen for their assistance in data analysis. We greatly appreciate all patients for their participat ion in this study. Author’s contributions Yan Gong designed the study and wrote the manuscript. Jesse Li-Ling contributed to revise the article. Hao Tan designed the experiments. Taiqing Zhong performed the experiments. Jiajing Wei and Dongsheng Xiong participated in sample collection. All authors have read and approved the final manuscript. Funding This stu dy was sponsored by the Scientific Research Pro gram of Sichuan Medical Association (No. S17060 ), the Technology Innovation Pro gram of the Science and Technology Bureau of Chengdu (No. 2018-YF05-00247-SN ), t he Scientific Research Project of Sichuan Provincial Health Commission (No. 20PJ123), Science and Technology Innovation Fund of Sichuan Provincial Hospital for Women and Children ( No. 20180205) and the Innovation and Entrepreneurship Training Program for College Students of China (No. 508-2043140 ). Availability of data and materials The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. Ethics approval and consent to participate Ethics approval was obtained from the Medical Ethics Committee of Sichuan Provincial Women ’ s and Children ’ s Hospital. Consent for publication Not applicable. Competing interests All authors have declared that they have no competing interests. Abbreviations GDF9 : Growth differentiation factor 9; BMP15 : B one morphogenetic protein 15; POR: P oor ovarian response; FF: F ollicle fluid; GCs: G ranulosa cells; IVF-ET: I n vitro fertilization and embryo transfer; ELISA: E nzyme - linked immunosorbent assay; qRT -PCR: Q uantitative real-time polymerase chain reaction; AFC: A ntral follicle count; CLBR: C umulative live birth rate; DOR: D ecreased ovarian reserve; OSFs: Oocyte-secreted factors; TGFβ: T ransforming growth factor β; POI: P remature ovarian insufficiency; COS: C ontrolled ovarian stimulation; ICSI: I ntracytoplasmic sperm injection; FSH: F ollicle stimulating hormone; hMG : human menopausal gonadotrop h in; u h CG : urinary human chorionic gonadotrophin; 2PN: T wo pronuclei; E 2 : E stradiol ; P: P rogesterone ; TT: T otal testosterone; PRL : P rolactin References Ferraretti AP, La Marca A, Fauser BC, Tarlatzis B, Nargund G, Gianaroli L, et al. 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Poseidon G, Alviggi C, Andersen CY, Buehler K, Conforti A, De Placido G, et al. A new more detailed stratification of low responders to ovarian stimulation: from a poor ovarian response to a low prognosis concept. Fertil Steril . 2016 ; 105:1452-3. Oudendijk JF, Yarde F, Eijkemans MJ, Broekmans FJ, Broer SL . The poor responder in IVF: is the prognosis always poor?: a systematic review. Hum Reprod Update . 2012 ; 18:1-11. Gonzalez-Foruria I, Penarrubia J, Borras A, Manau D, Casals G, Peralta S, et al. Age, independent from ovarian reserve status, is the main prognostic factor in natural cycle in vitro fertilization. Fertil Steril . 2016 ; 106:342-7. Younis JS, Ben-Ami M, Ben-Shlomo I . The Bologna criteria for poor ovarian response: a contemporary critical appraisal. J Ovarian Res . 2015 ; 8:76. Yang Y, Sun X, Cui L, Sheng Y, Tang R, Wei D, et al. Younger poor ovarian response women achieved better pregnancy results in the first three IVF cycles. Reprod Biomed Online . 2016 ; 32:532-7. Yakin K, Oktem O, Balaban B, Urman B . Bologna criteria are predictive for ovarian response and live birth in subsequent ovarian stimulation cycles. Arch Gynecol Obstet . 2019 ; 299:571-7. Xu B, Chen Y, Geerts D, Yue J, Li Z, Zhu G, et al. Cumulative live birth rates in more than 3,000 patients with poor ovarian response: a 15-year survey of final in vitro fertilization outcome. Fertil Steril . 2018 ; 109:1051-9. Kyrou D, Kolibianakis EM, Venetis CA, Papanikolaou EG, Bontis J, Tarlatzis BC . How to improve the probability of pregnancy in poor responders undergoing in vitro fertilization: a systematic review and meta-analysis. Fertil Steril . 2009 ; 91:749-66. Laissue P, Christin-Maitre S, Touraine P, Kuttenn F, Ritvos O, Aittomaki K, et al. Mutations and sequence variants in GDF9 and BMP15 in patients with premature ovarian failure. Eur J Endocrinol . 2006 ; 154:739-44. Otsuka F, McTavish KJ, Shimasaki S . Integral role of GDF-9 and BMP-15 in ovarian function. Mol Reprod Dev . 2011 ; 78:9-21. Wang TT, Ke ZH, Song Y, Chen LT, Chen XJ, Feng C, et al. Identification of a mutation in GDF9 as a novel cause of diminished ovarian reserve in young women. Hum Reprod . 2013, 28:2473-81. Chang HM, Qiao J, Leung PC . Oocyte-somatic cell interactions in the human ovary-novel role of bone morphogenetic proteins and growth differentiation factors. Hum Reprod Update . 2016 ; 23:1-18. Juengel JL, McNatty KP . The role of proteins of the transforming growth factor-beta superfamily in the intraovarian regulation of follicular development. Hum Reprod Update . 2005 ; 11:143-60. Russell DL, Robker RL . Molecular mechanisms of ovulation: co-ordination through the cumulus complex. Hum Reprod Update . 2007 ; 13:289-312. Fang L, Cheng JC, Chang HM, Sun YP, Leung PC . EGF-like growth factors induce COX-2-derived PGE2 production through ERK1/2 in human granulosa cells. J Clin Endocrinol Metab . 2013 ; 98:4932-41. Wu YT, Wang TT, Chen XJ, Zhu XM, Dong MY, Sheng JZ, et al. Bone morphogenetic protein-15 in follicle fluid combined with age may differentiate between successful and unsuccessful poor ovarian responders. Reprod Biol Endocrinol . 2012 ; 10:116. Li Y, Li RQ, Ou SB, Zhang NF, Ren L, Wei LN, et al. Increased GDF9 and BMP15 mRNA levels in cumulus granulosa cells correlate with oocyte maturation, fertilization, and embryo quality in humans. Reprod Biol Endocrinol . 2014 ; 12:81. Wu YT, Tang L, Cai J, Lu XE, Xu J, Zhu XM, et al. High bone morphogenetic protein-15 level in follicular fluid is associated with high quality oocyte and subsequent embryonic development. Hum Reprod . 2007 ; 22:1526-31. Alpha Scientists in Reproductive M, Embryology ESIGo . The Istanbul consensus workshop on embryo assessment: proceedings of an expert meeting. Hum Reprod . 2011 ; 26:1270-83. Haadsma ML, Groen H, Mooij TM, Burger CW, Broekmans FJ, Lambalk CB, et al. Miscarriage risk for IVF pregnancies in poor responders to ovarian hyperstimulation. Reprod Biomed Online . 2010 ; 20:191-200. Eichenlaub-Ritter U . Oocyte ageing and its cellular basis. Int J Dev Biol . 2012 ; 56:841-52. Shahine LK, Marshall L, Lamb JD, Hickok LR . Higher rates of aneuploidy in blastocysts and higher risk of no embryo transfer in recurrent pregnancy loss patients with diminished ovarian reserve undergoing in vitro fertilization. Fertil Steril . 2016, 106:1124-8. Franasiak JM, Forman EJ, Hong KH, Werner MD, Upham KM, Treff NR, et al. The nature of aneuploidy with increasing age of the female partner: a review of 15,169 consecutive trophectoderm biopsies evaluated with comprehensive chromosomal screening. Fertil Steril . 2014, 101:656-63. Hussein TS, Froiland DA, Amato F, Thompson JG, Gilchrist RB . Oocytes prevent cumulus cell apoptosis by maintaining a morphogenic paracrine gradient of bone morphogenetic proteins. J Cell Sci . 2005 ; 118:5257-68. Hobeika E, Armouti M, Kala H, Fierro MA, Winston NJ, Scoccia B, et al. Oocyte- s ecreted f actors s ynergize w ith FSH to p romote a romatase e xpression in p rimary h uman c umulus c ells. J Clin Endocrinol Metab . 2019 ; 104:1667-76. Sugiura K, Su YQ, Diaz FJ, Pangas SA, Sharma S, Wigglesworth K, et al. Oocyte-derived BMP15 and FGFs cooperate to promote glycolysis in cumulus cells. Development . 2007 ; 134:2593-603. Chang HM, Cheng JC, Klausen C, Leung PC . BMP15 suppresses progesterone production by down-regulating StAR via ALK3 in human granulosa cells. Mol Endocrinol . 2013 ; 27:2093-104. Su YQ, Wu X, O'Brien MJ, Pendola FL, Denegre JN, Matzuk MM, et al. Synergistic roles of BMP15 and GDF9 in the development and function of the oocyte-cumulus cell complex in mice: genetic evidence for an oocyte-granulosa cell regulatory loop. Dev Biol . 2004 ; 276:64-73. Li R, Albertini DF . The road to maturation: somatic cell interaction and self-organization of the mammalian oocyte. Nat Rev Mol Cell Biol . 2013 ; 14:141-52. Zhang HH, Xu PY, Wu J, Zou WW, Xu XM, Cao XY, et al. Dehydroepiandrosterone improves follicular fluid bone morphogenetic protein-15 and accumulated embryo score of infertility patients with diminished ovarian reserve undergoing in vitro fertilization: a randomized controlled trial. J Ovarian Res . 2014 ; 7:93. Barroso G, Barrionuevo M, Rao P, Graham L, Danforth D, Huey S, et al. Vascular endothelial growth factor, nitric oxide, and leptin follicular fluid levels correlate negatively with embryo quality in IVF patients. Fertil Steril . 1999 ; 72:1024-6. Younis JS, Laufer N . Recombinant luteinizing hormone supplementation to recombinant follicle stimulating hormone therapy in gonadotropin releasing hormone analogue cycles: what is the evidence? Curr Med Res Opin . 2018 ; 34:881-6. Crawford JL, McNatty KP . The ratio of growth differentiation factor 9: bone morphogenetic protein 15 mRNA expression is tightly co-regulated and differs between species over a wide range of ovulation rates. Mol Cell Endocrinol . 2012 ; 348:339-43. Christoforou ER, Pitman JL . Intrafollicular growth differentiation factor 9: bone morphogenetic 15 ratio determines litter size in mammalsdagger. Biol Reprod . 2019 ; 100:1333-43. Sudiman J, Ritter LJ, Feil DK, Wang X, Chan K, Mottershead DG, et al. Effects of differing oocyte-secreted factors during mouse in vitro maturation on subsequent embryo and fetal development. J Assist Reprod Genet . 2014 ; 31:295-306. Hussein TS, Thompson JG, Gilchrist RB . Oocyte-secreted factors enhance oocyte developmental competence. Dev Biol . 2006 ; 296:514-21. Supplementary Files coidisclosureDongshengXiong.pdf coidisclosureHaoTan.pdf coidisclosureTaiQingZhong.pdf coidisclosureJesseLiLing.pdf coidisclosureJiajingWei.pdf coidisclosureYanGong.pdf Cite Share Download PDF Status: Published Journal Publication published 04 Jan, 2021 Read the published version in Journal of Ovarian Research → Version 3 posted Editor assigned by journal 16 Dec, 2020 Editorial decision: Accept 16 Dec, 2020 Submission checks completed at journal 16 Dec, 2020 Editor invited by journal 16 Dec, 2020 You are reading this latest preprint version Show more versions 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. 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Also discoverable on Platform About Our Team In Review Editorial Policies 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-29352","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research","associatedPublications":[],"authors":[{"id":6808610,"identity":"15fb85e7-4311-4698-bc47-bc73f05cf2f9","order_by":0,"name":"Yan Gong","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA5ElEQVRIiWNgGAWjYBADHn5m5oMPEipqiNciJ9nOlmzw4Mwx4rUYG/TzmEk+bGEmrFTevffwa56aO4kbmHnMKhIb2Bj427sT8GoxPHMuzZrn2LPE7cxsZTcSd8gwSJw5uwG/lhk5ZsY8bIcTdzYzb7uReIaNwUAilxgt/w4nbjjMYFaQ2MZMWIu8RI7xY962w8YGh1nMGIjSYsBzxoxxbt9hOclmtmSJhDPHeAj6Rb69x/jDm2+Hefj5Dx/8+KOiRo6/vZeALQcY2KR4kAR4cCqF29LAwPzxB0Flo2AUjIJRMKIBADXQSxQxXC0AAAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0002-3562-971X","institution":"Chengdu Medical College","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Yan","middleName":"","lastName":"Gong","suffix":""},{"id":6808611,"identity":"c2c0fc63-815f-4fd0-9b55-8c8bb3d5668c","order_by":1,"name":"Jesse Li-Ling","email":"","orcid":"","institution":"Sichuan University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Jesse","middleName":"","lastName":"Li-Ling","suffix":""},{"id":6808612,"identity":"d4633cdc-b8d0-484d-8b28-8f66a7681385","order_by":2,"name":"Dongsheng Xiong","email":"","orcid":"","institution":"Chengdu Medical College","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Dongsheng","middleName":"","lastName":"Xiong","suffix":""},{"id":6808613,"identity":"207c09a7-12ce-42d4-97b4-be50a1fe9cef","order_by":3,"name":"Jiajing Wei","email":"","orcid":"","institution":"Chengdu Medical College","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Jiajing","middleName":"","lastName":"Wei","suffix":""},{"id":6808614,"identity":"8854ddfd-3ddb-4481-a24f-fe22cd019c80","order_by":4,"name":"Taiqing Zhong","email":"","orcid":"","institution":"Chengdu Medical College","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Taiqing","middleName":"","lastName":"Zhong","suffix":""},{"id":6808615,"identity":"27475a8e-9c0a-477b-bd13-961d48f24e0a","order_by":5,"name":"Hao Tan","email":"","orcid":"","institution":"Chengdu Medical College","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Hao","middleName":"","lastName":"Tan","suffix":""}],"badges":[],"createdAt":"2020-05-14 21:52:07","currentVersionCode":3,"declarations":"","doi":"10.21203/rs.3.rs-29352/v3","doiUrl":"https://doi.org/10.21203/rs.3.rs-29352/v3","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s13048-020-00757-x","type":"published","date":"2021-01-04T15:03:40+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":4721429,"identity":"0860be0d-8deb-481f-9150-1a7f65000635","added_by":"auto","created_at":"2021-01-05 17:12:46","extension":"tif","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":5503924,"visible":true,"origin":"","legend":"The gene and protein levels of GDF9 and BMP15 in GCs were decreased with increasing age. A The relatively mRNA levels of GDF9 and BMP15 were significantly different between the three groups (P \u003c 0.05). They were decreased with increasing age. GAPDH,β-actin and HPRT were used as the internal controls. B The relatively protein levels of GDF9 and BMP15 were significantly different between the three groups (P \u003c 0.05). They were decreased with increasing age. GAPDH was used as internal control. C A single band at 51 kDa represented GDF9, and 45 kDa represented BMP15 by western blot, respectively. 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Patients with POR have fewer\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003eretrieved oocytes\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e,\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003efewer\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;transferable embryos, lower pregnancy rates, and greater odds for cycle cancellation and miscarriage\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e[1]\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e.\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003eWith a\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;prevalence\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003eof\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;9%\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;~\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e24% in various IVF\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003ecenters\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e[2]\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e, POR\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;affects\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003eapproximately\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e11.9% of Chinese women undergoing IVF\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e-ET treatment\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e[3]\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e. Although\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003evarious\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;stimulation protocols\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003eand\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;strategies have been proposed,\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;patients with\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003ePOR\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;still bene\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003efi\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003et\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003eed\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;little from\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003esuch\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;treatment\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e[4]\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e.\u003c/span\u003e\u003c/p\u003e\n\u003cp\u003e\u003cspan data-contrast=\"none\"\u003eAccording to the Bologna criteria, POR\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003eis\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;diagnosed with at least two of the\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003efollowing\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003ethree criteria: advanced maternal age or any other risk factor for POR; previous history of POR; and an abnormal ovarian reserve test\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e[5]\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e. Although\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003eabove\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;criteria\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003eare\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;useful for predicting ovarian response and\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003ecounseling purpose, it\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003ehas\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003egrouped together women with various\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003eetiologies\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;and\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003eclinical\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003echaracteristics\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e[6]\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e. Some have reported that, compared with elder patients, younger poor ovarian responders (\u0026lt;\u0026thinsp;40, especially \u0026lt;\u0026thinsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e35 year old\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e)\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;have\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003ehigher\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;embryo quality and pregnancy rate but lower miscarriage rate\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e[7-9]\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e.\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003eA\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003ege has\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003etherefore\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003ebeen proposed as an independent predictor for POR\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e[7,8]\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e. Studies have shown that elder poor responders, in particular those over 40,\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003ehave\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;significantly lower cumulative live birth rate (CLBR) compared with young poor responders (\u0026lt; 35)\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e[10-12]\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e. Therefore, 35 and 40 year\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003es\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;old\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003ehave been\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003eus\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003eed as the thresholds by the Poseidon Criteria and Bologna Criteria, respectively\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e[5,6]\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e.\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003eFor\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003eelder poor responders\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e, a\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003ege-related infertility and decreased ovarian reserve (DOR) are the main obstacles, whereas\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003efor\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;younger poor responders, ovarian ag\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003eing seems to be\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;independent of\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003eage. The heterogeneous prognosis may be\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003eattributable to\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;various\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003eetiology of\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003ethe\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;patients.\u003c/span\u003e\u003c/p\u003e\n\u003cp\u003e\u003cspan data-contrast=\"none\"\u003eRisk factors for POR include fecundity decline with age, ovarian cystectomy, chronic smoking, genetic factors, previous chemotherapy and/or radiotherapy\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e, etc.\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e[13]\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e. The pathophysiology of POR is complex\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;and may involve\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;declined endocrine feedback of ovarian factors\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e[14-16]\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e.\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003eOocyte-secreted factors (OSFs) can regulate follicle development\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e[17]\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e.\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003eThe\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;transforming growth factor \u0026beta; (TGF\u0026beta;) superfamily is one of the most important OSFs which control the function of granulosa cells (GCs)\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e. The latter in turn\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;can nourish the oocyte\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e[18]\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e. Growth differentiation factor 9 (\u003c/span\u003e\u003cem\u003e\u003cspan data-contrast=\"none\"\u003eGDF9\u003c/span\u003e\u003c/em\u003e\u003cspan data-contrast=\"none\"\u003e) and bone morphogenetic protein 15 (\u003c/span\u003e\u003cem\u003e\u003cspan data-contrast=\"none\"\u003eBMP15\u003c/span\u003e\u003c/em\u003e\u003cspan data-contrast=\"none\"\u003e, also known as\u0026nbsp;\u003c/span\u003e\u003cem\u003e\u003cspan data-contrast=\"none\"\u003eGDF9\u003c/span\u003e\u003c/em\u003e\u003cem\u003e\u003cspan data-contrast=\"none\"\u003eB\u003c/span\u003e\u003c/em\u003e\u003cspan data-contrast=\"none\"\u003e) are essential members of the TGF-\u0026beta; superfamily\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;which can\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003eenhance\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;the proliferation and metabolism of\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003ethe\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003eGCs and stimulat\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003ee\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;the expression of kit ligand (KL)\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003ein\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;GCs.\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003eThe\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003eGCs\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003ecan\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003ethen provide nutrients, steroid hormones and growth factors\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003ewhich can\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;enable\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003eoocyte maturation via\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003ean\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003eintimate cross-talk\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e[15,17]\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e. Cumulus expansion is a compl\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003eex\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;process which is\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003ecritical\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;for oocyte maturation and ovulation\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e[19]\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e. The process\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003eis\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;highly dependent on the interactions of two signals\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003ereleased\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;by epidermal growth factor (EGF)-like peptides and OSFs\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e[20]\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e. Previous studies have shown that genetic variants and abnormal expression of\u0026nbsp;\u003c/span\u003e\u003cem\u003e\u003cspan data-contrast=\"none\"\u003eGDF9\u003c/span\u003e\u003c/em\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;and\u0026nbsp;\u003c/span\u003e\u003cem\u003e\u003cspan data-contrast=\"none\"\u003eBMP15\u003c/span\u003e\u003c/em\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;may\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003epredispose to\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;follicle atresia and early exhaustion of ovarian reserve\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e[15,16]\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e. Altered expression of\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003ethe two\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;have been found in patients with premature ovarian insufficiency (POI), DOR and infertility\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e[14-16,21]\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e. Moreover, for patients undergoing IVF-ET treatment,\u0026nbsp;\u003c/span\u003e\u003cem\u003e\u003cspan data-contrast=\"none\"\u003eGDF9\u003c/span\u003e\u003c/em\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;and\u0026nbsp;\u003c/span\u003e\u003cem\u003e\u003cspan data-contrast=\"none\"\u003eBMP15\u003c/span\u003e\u003c/em\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;in follicular fluid (FF) and GCs have been associated with poorer oocyte quality and outcome\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;of treatment\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e[22]\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e, hence\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;were proposed as biomarkers for the potential of oocyte development\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e[22,23]\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e.\u003c/span\u003e\u003c/p\u003e\n\u003cp\u003e\u003cspan data-contrast=\"none\"\u003eConsidering the important roles of\u0026nbsp;\u003c/span\u003e\u003cem\u003e\u003cspan data-contrast=\"none\"\u003eGDF9\u003c/span\u003e\u003c/em\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;and\u0026nbsp;\u003c/span\u003e\u003cem\u003e\u003cspan data-contrast=\"none\"\u003eBMP15\u003c/span\u003e\u003c/em\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;in\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003efolliculogenesis\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e, this\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003eprospective study was designed to analyze the expression of\u0026nbsp;\u003c/span\u003e\u003cem\u003e\u003cspan data-contrast=\"none\"\u003eGDF9\u003c/span\u003e\u003c/em\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;and\u0026nbsp;\u003c/span\u003e\u003cem\u003e\u003cspan data-contrast=\"none\"\u003eBMP15\u003c/span\u003e\u003c/em\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;in the\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003eFF\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003eand GCs from patients with POR from\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003emore homogeneous age groups\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e.\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003eIdentification of the underlying mechanism may facilitate design of individualized treatment protocols for subgroups of patients with POR.\u003c/span\u003e\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cstrong\u003e\u003cspan data-contrast=\"none\"\u003ePopulation\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cspan data-contrast=\"none\"\u003eThis study was registered with the\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003eChinese Clinical Trial Registry Center (Registration No. ChiCTR1800016107) a\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003end approv\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003eed\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;by the Medical Ethics Committee of Sichuan Provincial\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003eWomen\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026rsquo;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003es and Children\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026rsquo;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003es\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;Hospital\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e. All patients had given written informed consent.\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003eThe study\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003ealso\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003econformed to the Declaration of Helsinki for Medical Research involving Human Subjects (2013 revision).\u003c/span\u003e\u003c/p\u003e\n\u003cp\u003e\u003cspan data-contrast=\"none\"\u003eEthnic Han Chinese patients diagnosed with POR\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003eby\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;the Bologna criteria\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e[1]\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;were enrolled from May 2018\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;to\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003eOctober 2019 and divided into\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003ethree groups:\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003egroup A (\u0026lt;\u0026thinsp;35 year old), group B (35\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e-\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e40 year old), and group C (\u0026gt; 40 year old). All patients underwent IVF-ET treatment.\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003ePatients were excluded from the study\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003eshould\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;they me\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003ee\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003et any of\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003ethe following criteria: (1)\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003eC\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003eongenital\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;uterine malformation, endometriosis,\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003epolycystic ovarian syndrome,\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003eintrauterine adhesion, single ovary;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;(2) S\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003eystemic\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;lupus erythematosus and/or sicca syndrome;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;(3) U\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003encontrolled\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;endocrinopathy such as diabetes, hyper\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e/\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003ehypothyroidism, and hyperprolactinemia;\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e(4) A\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003ebnormal\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003echromosomal\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003ekaryotype\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;(5)\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003eC\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003eontrolled\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;ovarian stimulation (COS) in\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003ethe\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003epast three months; and (6)\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003eI\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003entracytoplasmic\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;sperm injection (ICSI) cycle\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003edue to\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;male factor infertility.\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003eMedical\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003ehis\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003etory\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003ew\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003eas\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003etaken for\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;all participants, including\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003eregularity of\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003emenstrual cycle, duration of infertility and pre-treatment protocols.\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003eH\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003eeight and body weight (with shoes and heavy clothing\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;taken off\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e) were measured. Body weight index (BMI) was calculated as weight divided by height squared (kg/m\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u003cspan data-fontsize=\"10.5\"\u003e2\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e).\u003c/span\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cspan data-contrast=\"none\"\u003eCOS\u003c/span\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;and IVF procedures\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cspan data-contrast=\"none\"\u003eA GnRH antagonist protocol was conducted for all patients.\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;F\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003erom day 2 or 3 of the cycle, 187.5\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;~\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e225\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;IU/d\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;human recombinant follicle stimulating hormone (\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003erFSH\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e) (\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003eGonal\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e-F, Merck Serono, Germany;\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003ePuregon\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026reg;, Merck Sharp \u0026amp; Dohme, USA) and 75\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;IU/d\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003ehuman menopausal\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003egonadotrop\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003eh\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003ein (\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003ehMG\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e) (\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003emenotrophin\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e,\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003eLivzon\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;Pharmaceutical Group Inc, Zhuhai, China\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e) were injected. The dosage of\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003erFSH\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;and\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003ehMG\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;was adjusted according to\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003ethe\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003eovarian response. Ganirelix\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e(\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003eMerck Sharp \u0026amp; Dohme, USA\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e) was administered should one of the following criteria be met: serum estradiol (E\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u003cspan data-fontsize=\"10.5\"\u003e2\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e) \u0026gt; 300\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003epg\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e/ml, follicle diameter \u0026gt; 14 mm,\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003eluteinizing hormone (LH)\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;\u0026gt; 10 IU/L\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e. When the leading follicle reached 18 mm in diameter, 10 000 IU of urinary human chorionic gonadotrophin (\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003eu\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003eh\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003eCG\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e) (\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003eLivzon\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;Pharmaceutical Group Inc, Zhuhai, China) was injected to trigger the ovulation.\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;Oocytes were retrieved by trans-vaginal ultrasound guidance within approximately 36 h after\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;the\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;trigger, and follicle flushing was not\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003eused\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e.\u003c/span\u003e\u003c/p\u003e\n\u003cp\u003e\u003cspan data-contrast=\"none\"\u003eOocytes were fertilized by conventional IVF for 4\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;~\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e6 h\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e.\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003eMature\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;oocyte was defined as being at the metaphase\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003eⅡ\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;(\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003eMⅡ\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e) stage with the first polar body visible in the cytoplasm. 17\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;~\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e18 h after the IVF, normal fertilized oocyte was confirmed\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003eshould\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;it contain two pronuclei (2PN). Cultured embryos were evaluated on day 3 based on the number of blastomeres and degree of fragmentation\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e. Embryos of grade A\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;~\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003eC on day 3 were defined as transplantable embryos\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e[24]\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e.\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003eOne or two\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003etransplantable\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;embryos were transferred. Luteal phase support was started on the oocyte retrieval day\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003ewith\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;the injection of 60 mg/d progesterone oil (Zhejiang\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003eXianju\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;Pharmaceutical Co., Ltd. Taizhou, China) or vaginal progesterone (\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003eCrinone\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;8% gel, Merck, Germany).\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003eThe reasons for c\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003eancel\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003eed\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;cycle\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003es\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;included follicular growth failure (10 days after COS, leading follicle diameter \u0026lt; 10\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003emm), failed oocyte retrieval at the time of follicle aspiration,\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003eno transplantable embryos (no\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003emature oocyte, abnormal fertilization or cleavage), and\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003eaccumulation of embryos and progesterone \u0026gt; 2.5 ng/ml on the trigger day.\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003eC\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003elinical\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;pregnancy was defined as\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003edetection\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;of embryonic heartbeat.\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003eThe r\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003eates of implantation, clinical pregnancy, multiple pregnancy, and miscarriage were calculated\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e[8]\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e.\u003c/span\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cspan data-contrast=\"none\"\u003eMeasurement of basal endocrine parameters in serum\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cspan data-contrast=\"none\"\u003eE\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003endocrine\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;parameters including E\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u003cspan data-fontsize=\"10.5\"\u003e2\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e, progesterone (P), total testosterone (TT), prolactin (PRL), FSH and\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003eLH\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003ewere measured\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003ewith an\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;electrochemiluminescence immunoassay platform (Roche Diagnostics GmbH, Mannheim, Germany)\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e.\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;Anti-M\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026uuml;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003ellerian\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;hormone (AMH) was measured\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003ewith an\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;enzyme\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e-\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003elinked immunosorbent assay kit (Guangzhou\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003eKangrun\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;Biotech, Co., Ltd, Guangdong, China).\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003eI\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003entra\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e- and inter-assay coefficients\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003efor the\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;above varia\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003eble\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003es were\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003eset as\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026lt; 5% and 10%, respectively.\u003c/span\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cspan data-contrast=\"none\"\u003eCollection of FF and GCs\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cspan data-contrast=\"none\"\u003eFF samples were carefully collected from follicles with a diameter\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003eof\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026ge;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e18 mm,\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003eand\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003ecentrifug\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003eed\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003eimmediately at 700\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026times;\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003eg for 5 min\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e. T\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003ehe supernatant was stored at -80\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e℃.\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003eGCs were obtained by follicular aspiration and isolated from blood cells and cellular debris\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003ewith a\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003elymphocyte separation medium (\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003eBeijing\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003eSolarbio\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;Science\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;and\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003eTechnology Corporation,\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003eBeijing,\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003eChina) by centrifugation at 700\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026times;\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003eg\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;for 10 min.\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003eR\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003eesidual\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;red blood cells were removed\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003ewith a\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;red blood cell lysis buffer (\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003eSolarbio\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;Science\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;and\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003eTechnology Corporation\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e,\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;Beijing, China\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e). The\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003eGCs were stored at\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e-\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e80℃ until the time of use.\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;For each patient, the FF and\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003eGCs were collected\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003efrom all follicles\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003eand\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003epooled as\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;one\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003esample.\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cspan data-contrast=\"none\"\u003eDetermination of\u0026nbsp;\u003c/span\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cspan data-contrast=\"none\"\u003eGDF9 and BMP15 in FF by\u0026nbsp;\u003c/span\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cspan data-contrast=\"none\"\u003eELISA\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cspan data-contrast=\"none\"\u003eThe concentrations of GDF9 and BMP15 in FF were measured with a commercial enzyme-linked immunosorbent assay kit (\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003eElabscience\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;Biotechnology Co., Ltd., Wuhan, China) by following the manufacturer\u0026rsquo;s instructions.\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003eThe standard product in GDF9 assay kit\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003ewas\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;human GDF9 recombinant protein expressed in\u003c/span\u003e\u003cem\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;E. coli\u003c/span\u003e\u003c/em\u003e\u003cspan data-contrast=\"none\"\u003e, and the sequence\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003ewas\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;Gly320-Arg\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e454. The kit has been crossed experimented with human GDF-2, GDF-7, GDF-10, GDF-11, GDF-15 and BMP-15\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e, with t\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003ehe crossov\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003eer rates\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003ebeing\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;0.74%, 0.61%, 0.78%, 0.65%, 0.72% and 0.41%, respectively. The standard product\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003eof the\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;BMP15 assay kit\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003ewa\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003es human BMP15 recombinant protein expressed in\u0026nbsp;\u003c/span\u003e\u003cem\u003e\u003cspan data-contrast=\"none\"\u003eE. coli\u003c/span\u003e\u003c/em\u003e\u003cspan data-contrast=\"none\"\u003e, and the sequence\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003ewas\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;Gln268-Arg392. The kit has been crossed experimented with human BMP-1, BMP-2, BMP-4, BMP-6, BMP-7 and GDF9\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e, with the\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;crossover rates\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003ebeing\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;0.53%, 0.92%, 0.79%, 0.56%, 0.74% and 0.38%, respectively.\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;The standard\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003euse of\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;the two kits\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;was to make serial dilutions, i.e., 1000, 500, 250, 125, 62.5, 31.25, 15.63, 0\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003epg\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e/ml, of a working solution of 1000\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003epg\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e/ml before the measurement. The FF samples were thawed and mixed together,\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003eand\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e100\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026mu;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003el\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;FF was\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003eadded to each well in duplicate. The absorbance value was measured at 450 nm with a\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003ePerlong\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;DNM-9602G microplate spectrophotometer (\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003ePerlong\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;New Technology Co., Ltd., Beijing, China).\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003eThe sensitivity of the assay was set as 9.38\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003epg\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e/ml.\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003eThe detection range of GDF9 and BMP15 w\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003eas\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;set as 15.36 ~ 1000\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003epg\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e/ml. The concentration of GDF in some FF samples were above the range. Such samples were diluted 2 times,\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003eand\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;100\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026mu;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003el\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;diluted FF was\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003ethen\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003eadded\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003eto\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;each well. The final concentration was\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003ecalculated\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;by\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003emultipl\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003eying\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;the detection value\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003ewith\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003ethe dilution factor.\u003c/span\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cspan data-contrast=\"none\"\u003eDetermination of mRNA expression in GCs by\u0026nbsp;\u003c/span\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cspan data-contrast=\"none\"\u003equantitative real-time polymerase chain reaction (\u003c/span\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cspan data-contrast=\"none\"\u003eqRT\u003c/span\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cspan data-contrast=\"none\"\u003e-PCR)\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cspan data-contrast=\"none\"\u003eTotal RNA was isolated from GCs\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003ewith\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;a\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003eRNAprep\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;Pure Micro Kit (\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003eTiangen\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;Biotech Co., Ltd., Beijing, China). The\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003epurity and concentration of RNA were determined with Nanodrop-2000 (Thermo Fisher Scientific,\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003eWaltham, Massachusetts, USA)\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003eunder\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;the absorbance of 260 nm/280 nm. The RNA was reversely transcribed into cDNA using a\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003ePrimeScript\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u003cspan data-fontsize=\"10.5\"\u003eTM\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;RT Reagent Kit with gDNA Eraser (\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003eTaKaRa\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e, Tokyo, Japan). The cDNA was then amplified using\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003eTB\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003eGreen\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u003cspan data-fontsize=\"10.5\"\u003eTM\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;Premix Ex\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003eTaq\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u003cspan data-fontsize=\"10.5\"\u003eTM\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;Ⅱ (\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003eTaKaRa\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e, Tokyo, Japan) by\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003eqRT\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e-PCR in triplicate.\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003eThe\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003ePCR conditions were 95℃ for 30 s, followed by 40 cycles of 95℃ for 10 s, 60℃ for 30 s, and 65℃ for 5 s. Formation of a single product was verified with a melting curve method.\u003c/span\u003e\u003cem\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;GAPDH\u003c/span\u003e\u003c/em\u003e\u003cspan data-contrast=\"none\"\u003e,\u003c/span\u003e\u003cem\u003e\u003cspan data-contrast=\"none\"\u003e\u0026beta;-ac\u003c/span\u003e\u003c/em\u003e\u003cem\u003e\u003cspan data-contrast=\"none\"\u003etin\u003c/span\u003e\u003c/em\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;and\u0026nbsp;\u003c/span\u003e\u003cem\u003e\u003cspan data-contrast=\"none\"\u003eHPRT\u0026nbsp;\u003c/span\u003e\u003c/em\u003e\u003cspan data-contrast=\"none\"\u003ewere used as\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003einternal controls\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e.\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;The mRNA levels of the target genes were calculated\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003ewith\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;the 2\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u003cspan data-fontsize=\"10.5\"\u003e-\u0026Delta;CT\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;method and expressed as fold change relative to the geometric means of the three\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003einternal\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;controls.\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003eAll PCR reactions were\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003econducted in triplicate\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e.\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003ePrimers used in the\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003eqRT\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e-PCR are shown in Table 1.\u003c/span\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cspan data-contrast=\"none\"\u003eTable 1 Sequences of primers used in\u0026nbsp;\u003c/span\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cspan data-contrast=\"none\"\u003eqRT\u003c/span\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cspan data-contrast=\"none\"\u003e-PCR\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"1\" data-table=\"\" data-tablelook=\"0\"\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd data-celllook=\"4368\"\u003e\n\u003cp\u003e\u003cspan data-contrast=\"none\"\u003eGene\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd data-celllook=\"272\"\u003e\n\u003cp\u003e\u003cspan data-contrast=\"none\"\u003ePrimer (5'\u0026rarr;3')\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd data-celllook=\"272\"\u003e\n\u003cp\u003e\u003cspan data-contrast=\"none\"\u003eAnnealing temperature (℃)\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd data-celllook=\"272\"\u003e\n\u003cp\u003e\u003cspan data-contrast=\"none\"\u003eEfficienc\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003ey\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd data-celllook=\"4368\"\u003e\n\u003cp\u003e\u003cem\u003e\u003cspan data-contrast=\"none\"\u003eGDF9\u003c/span\u003e\u003c/em\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd data-celllook=\"272\"\u003e\n\u003cp\u003e\u003cspan data-contrast=\"none\"\u003eF: TGGAGCATCCTTCAGCAC\u003c/span\u003e\u003c/p\u003e\n\u003cp\u003e\u003cspan data-contrast=\"none\"\u003eR: GCAGCCTCTTCTCCCACA\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd data-celllook=\"272\"\u003e\n\u003cp\u003e\u003cspan data-contrast=\"none\"\u003e57.2\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd data-celllook=\"272\"\u003e\n\u003cp\u003e\u003cspan data-contrast=\"none\"\u003e9\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e8.8\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e%\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd data-celllook=\"4369\"\u003e\n\u003cp\u003e\u003cem\u003e\u003cspan data-contrast=\"none\"\u003eBMP15\u003c/span\u003e\u003c/em\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd data-celllook=\"273\"\u003e\n\u003cp\u003e\u003cspan data-contrast=\"none\"\u003eF: TTTACCGCCATCATCTCCAA\u003c/span\u003e\u003c/p\u003e\n\u003cp\u003e\u003cspan data-contrast=\"none\"\u003eR: TTTCCAAGCGTTAGACATCA\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd data-celllook=\"272\"\u003e\n\u003cp\u003e\u003cspan data-contrast=\"none\"\u003e53.4\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd data-celllook=\"272\"\u003e\n\u003cp\u003e\u003cspan data-contrast=\"none\"\u003e9\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e1.9\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e%\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd data-celllook=\"4369\"\u003e\n\u003cp\u003e\u003cem\u003e\u003cspan data-contrast=\"none\"\u003eGAPDH\u003c/span\u003e\u003c/em\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd data-celllook=\"273\"\u003e\n\u003cp\u003e\u003cspan data-contrast=\"none\"\u003eF: ACGGATTTGGTCGTATTGGG\u003c/span\u003e\u003c/p\u003e\n\u003cp\u003e\u003cspan data-contrast=\"none\"\u003eR: CGCTCCTGGAAGATGGTGAT\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd data-celllook=\"273\"\u003e\n\u003cp\u003e\u003cspan data-contrast=\"none\"\u003e57.4\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd data-celllook=\"273\"\u003e\n\u003cp\u003e\u003cspan data-contrast=\"none\"\u003e1\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e01.6\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e%\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd data-celllook=\"4369\"\u003e\n\u003cp\u003e\u003cem\u003e\u003cspan data-contrast=\"none\"\u003e\u0026beta;\u003c/span\u003e\u003c/em\u003e\u003cem\u003e\u003cspan data-contrast=\"none\"\u003e-actin\u003c/span\u003e\u003c/em\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd data-celllook=\"273\"\u003e\n\u003cp\u003e\u003cspan data-contrast=\"none\"\u003eF:\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003eGACAGGATGCAGAAGGAGAT\u003c/span\u003e\u003c/p\u003e\n\u003cp\u003e\u003cspan data-contrast=\"none\"\u003eR:\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003eCTGCTTGCTGATCCACATCT\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd data-celllook=\"273\"\u003e\n\u003cp\u003e\u003cspan data-contrast=\"none\"\u003e5\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e3.5\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd data-celllook=\"273\"\u003e\n\u003cp\u003e\u003cspan data-contrast=\"none\"\u003e99.6%\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd data-celllook=\"4369\"\u003e\n\u003cp\u003e\u003cem\u003e\u003cspan data-contrast=\"none\"\u003eH\u003c/span\u003e\u003c/em\u003e\u003cem\u003e\u003cspan data-contrast=\"none\"\u003ePRT\u003c/span\u003e\u003c/em\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd data-celllook=\"273\"\u003e\n\u003cp\u003e\u003cspan data-contrast=\"none\"\u003eF: CCATTCCTATGACTGTAGAT\u003c/span\u003e\u003c/p\u003e\n\u003cp\u003e\u003cspan data-contrast=\"none\"\u003eR: CCAGTTAAAGTTGAGAGATCAT\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd data-celllook=\"273\"\u003e\n\u003cp\u003e\u003cspan data-contrast=\"none\"\u003e5\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e1.8\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd data-celllook=\"273\"\u003e\n\u003cp\u003e\u003cspan data-contrast=\"none\"\u003e96.5%\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cspan data-contrast=\"none\"\u003eDetermination of\u0026nbsp;\u003c/span\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cspan data-contrast=\"none\"\u003eGDF9 and BMP15\u003c/span\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;in GCs by Western blotting\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cspan data-contrast=\"none\"\u003eGCs were lysed in RIPA lysis buffer (\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003eKeyGen\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;Biotech Co., Ltd., Nanjing, China) containing the\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003eHalt\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u003cspan data-fontsize=\"10.5\"\u003eTM\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;Protease Inhibitor Cocktail (Invitrogen, Karlsruhe, Germany).\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003eA\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003eBCA protein quantitative kit (Pierce, Thermo Fisher Scientific, Rockford, IL, USA)\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;was used\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003efor\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;determin\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003eing\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;the protein concentration.\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003eThe total proteins (60 \u0026micro;g/lane) were subjected to 10% SDS-PAGE and transferred onto a polyvinylidene fluoride (PVDF) membrane (EMD Millipore, Billerica, MA, USA).\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003eThe membrane was then blocked in TBST (Tris-buffered saline with Tween-20) containing 5% BSA (Bio-Rad, Hercules, CA, USA) at room temperature for 1 h, and incubated with primary antibody overnight at 4\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e℃\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e.\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003eThe p\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003erimary antibodies used in this study\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003ehave\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003einclude\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003ed GDF9 (1:500,\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003eBeyotime\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;Biotechnology Co., Ltd., Shanghai, China), BMP15 (1:500,\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003eBeyotime\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;Biotechnology Co., Ltd.),\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003eand GAPDH (1:2000,\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003eBioss\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e, Beijing, China).\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;After washed with TBST for three times, the membranes were incubated with secondary antibodies for 2 h at room temperature. The protein was then detected with a\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003eSuperSignal\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026reg; West Pico Trial Kit (Thermo Scientific Pierce, IL, USA). Band intensity was measured with a Gel Doc XR densitometer (Bio-Rad, Hercules, CA, USA) and normalized with that of the internal control.\u003c/span\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cspan data-contrast=\"none\"\u003eStatistical analysis\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cspan data-contrast=\"none\"\u003eData were analyzed\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003ewith\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;SPSS 17.0 software (SPSS Inc., Chicago IL, USA). Continuous variables were expressed as mean \u0026plusmn; standard deviation (SD). The Kolmogorov-Smirnov test\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003ewas used to assess the normality of data distribution. Continuous variables with normal distribution were compared\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003eusing one-way ANOVA with\u0026nbsp;\u003c/span\u003e\u003cem\u003e\u003cspan data-contrast=\"none\"\u003epost hoc\u003c/span\u003e\u003c/em\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;Bonferroni test\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e. Categorical data were compared using Chi-squared test.\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003ePearson\u0026rsquo;s correlation was used to assess the correlation between the relative mRNA levels in GCs and protein concentration\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003es\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;in FF.\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003eSignificance level was set as\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026lt;\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e0.05, and two-tailed test was used for all hypothesis tests.\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003eLinear regression equation\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003ewas\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;established for the expression levels of GDF9 against BMP15 mRNA in each sample.\u003c/span\u003e\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003e\u003cspan data-contrast=\"none\"\u003eBaseline characteristics of the patients\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cspan data-contrast=\"none\"\u003eBaseline characteristics of the patient are shown in Table 2. The average age\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003es\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;of the three groups\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003ehave differed\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;significantly (\u003c/span\u003e\u003cem\u003e\u003cspan data-contrast=\"none\"\u003eP\u0026nbsp;\u003c/span\u003e\u003c/em\u003e\u003cspan data-contrast=\"none\"\u003e\u0026lt; 0.05). Compared with group A, the duration of infertility was significantly longer in group C (\u003c/span\u003e\u003cem\u003e\u003cspan data-contrast=\"none\"\u003eP\u0026nbsp;\u003c/span\u003e\u003c/em\u003e\u003cspan data-contrast=\"none\"\u003e\u0026lt; 0.05). The abnormal menstrual cycle rate, BMI, AFC, AMH and basal hormone levels\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003edid not significantly differ among the three groups (\u003c/span\u003e\u003cem\u003e\u003cspan data-contrast=\"none\"\u003eP\u003c/span\u003e\u003c/em\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;\u0026gt; 0.05).\u003c/span\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cspan data-contrast=\"none\"\u003eTable 2 Baseline characteristics of the patients\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"1\" data-table=\"\" data-tablelook=\"1184\"\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd data-celllook=\"273\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd data-celllook=\"257\"\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cspan data-contrast=\"none\"\u003egroup A\u0026nbsp;\u003c/span\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cspan data-contrast=\"none\"\u003e(n = 63)\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd data-celllook=\"257\"\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cspan data-contrast=\"none\"\u003eGroup B (n = 65)\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd data-celllook=\"273\"\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cspan data-contrast=\"none\"\u003eGroup C (n = 68)\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd data-celllook=\"273\"\u003e\n\u003cp\u003e\u003cspan data-contrast=\"none\"\u003eAge (years)\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u003cspan data-fontsize=\"10.5\"\u003eabc\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd data-celllook=\"257\"\u003e\n\u003cp\u003e\u003cspan data-contrast=\"none\"\u003e30.65 \u0026plusmn; 2.57\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd data-celllook=\"257\"\u003e\n\u003cp\u003e\u003cspan data-contrast=\"none\"\u003e37.37\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026plusmn; 1.53\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd data-celllook=\"273\"\u003e\n\u003cp\u003e\u003cspan data-contrast=\"none\"\u003e42.59\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026plusmn;\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e1.70\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd data-celllook=\"273\"\u003e\n\u003cp\u003e\u003cspan data-contrast=\"none\"\u003eAbnormal menstrual cycle\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd data-celllook=\"257\"\u003e\n\u003cp\u003e\u003cspan data-contrast=\"none\"\u003e34.92% (22/63)\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd data-celllook=\"257\"\u003e\n\u003cp\u003e\u003cspan data-contrast=\"none\"\u003e36.92% (24/65)\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd data-celllook=\"257\"\u003e\n\u003cp\u003e\u003cspan data-contrast=\"none\"\u003e35.29% (24/68)\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd data-celllook=\"273\"\u003e\n\u003cp\u003e\u003cspan data-contrast=\"none\"\u003eDuration of infertility (\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003eyear\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003es)\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u003cspan data-fontsize=\"10.5\"\u003eb\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd data-celllook=\"4369\"\u003e\n\u003cp\u003e\u003cspan data-contrast=\"none\"\u003e3.32 \u0026plusmn; 2.21\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd data-celllook=\"4369\"\u003e\n\u003cp\u003e\u003cspan data-contrast=\"none\"\u003e4.03 \u0026plusmn; 3.05\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd data-celllook=\"4369\"\u003e\n\u003cp\u003e\u003cspan data-contrast=\"none\"\u003e4.68 \u0026plusmn; 3.00\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd data-celllook=\"273\"\u003e\n\u003cp\u003e\u003cspan data-contrast=\"none\"\u003eBMI (kg/m\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u003cspan data-fontsize=\"10.5\"\u003e2\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e)\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd data-celllook=\"257\"\u003e\n\u003cp\u003e\u003cspan data-contrast=\"none\"\u003e22.08\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026plusmn;\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e2.62\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd data-celllook=\"257\"\u003e\n\u003cp\u003e\u003cspan data-contrast=\"none\"\u003e22.21 \u0026plusmn;\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e2.00\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd data-celllook=\"273\"\u003e\n\u003cp\u003e\u003cspan data-contrast=\"none\"\u003e22.44\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026plusmn;\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e2.62\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd data-celllook=\"273\"\u003e\n\u003cp\u003e\u003cspan data-contrast=\"none\"\u003eAFC\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd data-celllook=\"257\"\u003e\n\u003cp\u003e\u003cspan data-contrast=\"none\"\u003e4.05 \u0026plusmn; 1.43\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd data-celllook=\"257\"\u003e\n\u003cp\u003e\u003cspan data-contrast=\"none\"\u003e3.91 \u0026plusmn; 1.69\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd data-celllook=\"273\"\u003e\n\u003cp\u003e\u003cspan data-contrast=\"none\"\u003e3.94 \u0026plusmn; 1.68\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd data-celllook=\"273\"\u003e\n\u003cp\u003e\u003cspan data-contrast=\"none\"\u003eFSH/LH ratio\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd data-celllook=\"257\"\u003e\n\u003cp\u003e\u003cspan data-contrast=\"none\"\u003e2.68\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026plusmn;\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e1.29\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd data-celllook=\"257\"\u003e\n\u003cp\u003e\u003cspan data-contrast=\"none\"\u003e2.76\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026plusmn;\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e1.71\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd data-celllook=\"273\"\u003e\n\u003cp\u003e\u003cspan data-contrast=\"none\"\u003e2.99\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026plusmn;\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e1.21\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd data-celllook=\"273\"\u003e\n\u003cp\u003e\u003cspan data-contrast=\"none\"\u003eE\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u003cspan data-fontsize=\"10.5\"\u003e2\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;(\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003epg\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e/ml\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e)\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd data-celllook=\"257\"\u003e\n\u003cp\u003e\u003cspan data-contrast=\"none\"\u003e76.12\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026plusmn;\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e34.71\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd data-celllook=\"257\"\u003e\n\u003cp\u003e\u003cspan data-contrast=\"none\"\u003e77.49\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026plusmn;\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e52.67\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd data-celllook=\"273\"\u003e\n\u003cp\u003e\u003cspan data-contrast=\"none\"\u003e85.08\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026plusmn;\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e50.88\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd data-celllook=\"273\"\u003e\n\u003cp\u003e\u003cspan data-contrast=\"none\"\u003eP\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;(\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003eng/ml\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e)\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd data-celllook=\"257\"\u003e\n\u003cp\u003e\u003cspan data-contrast=\"none\"\u003e0.77\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026plusmn; 0\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e.35\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd data-celllook=\"257\"\u003e\n\u003cp\u003e\u003cspan data-contrast=\"none\"\u003e0.78\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026plusmn; 0\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e.33\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd data-celllook=\"273\"\u003e\n\u003cp\u003e\u003cspan data-contrast=\"none\"\u003e0.84\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026plusmn; 0\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e.52\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd data-celllook=\"273\"\u003e\n\u003cp\u003e\u003cspan data-contrast=\"none\"\u003eTT\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;(\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003eng/ml\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e)\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd data-celllook=\"257\"\u003e\n\u003cp\u003e\u003cspan data-contrast=\"none\"\u003e0.37\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026plusmn; 0\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e.15\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd data-celllook=\"257\"\u003e\n\u003cp\u003e\u003cspan data-contrast=\"none\"\u003e0.36\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026plusmn; 0\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e.15\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd data-celllook=\"4369\"\u003e\n\u003cp\u003e\u003cspan data-contrast=\"none\"\u003e0.39\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026plusmn; 0\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e.10\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd data-celllook=\"273\"\u003e\n\u003cp\u003e\u003cspan data-contrast=\"none\"\u003eAMH\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;(\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003eng/ml\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e)\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd data-celllook=\"257\"\u003e\n\u003cp\u003e\u003cspan data-contrast=\"none\"\u003e0.80\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026plusmn;\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e0.39\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd data-celllook=\"257\"\u003e\n\u003cp\u003e\u003cspan data-contrast=\"none\"\u003e0.78\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026plusmn;\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e0.24\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd data-celllook=\"273\"\u003e\n\u003cp\u003e\u003cspan data-contrast=\"none\"\u003e0.73\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026plusmn;\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e0.39\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd data-celllook=\"273\"\u003e\n\u003cp\u003e\u003cspan data-contrast=\"none\"\u003ePRL\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;(\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003eng/ml\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e)\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd data-celllook=\"257\"\u003e\n\u003cp\u003e\u003cspan data-contrast=\"none\"\u003e199.84 \u0026plusmn; 39.35\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd data-celllook=\"257\"\u003e\n\u003cp\u003e\u003cspan data-contrast=\"none\"\u003e205.58 \u0026plusmn; 58.49\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd data-celllook=\"273\"\u003e\n\u003cp\u003e\u003cspan data-contrast=\"none\"\u003e197.26 \u0026plusmn; 59.45\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cspan data-contrast=\"none\"\u003eData are presented as mean \u0026plusmn; SD\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;or\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003epercentage (number).\u0026nbsp;\u003c/span\u003e\u003cstrong\u003e\u003cem\u003e\u003cspan data-contrast=\"none\"\u003eAbbreviations:\u0026nbsp;\u003c/span\u003e\u003c/em\u003e\u003c/strong\u003e\u003cem\u003e\u003cspan data-contrast=\"none\"\u003eBMI\u0026nbsp;\u003c/span\u003e\u003c/em\u003e\u003cspan data-contrast=\"none\"\u003eBody mass index,\u0026nbsp;\u003c/span\u003e\u003cem\u003e\u003cspan data-contrast=\"none\"\u003eAFC\u003c/span\u003e\u003c/em\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;Antral follicle count,\u0026nbsp;\u003c/span\u003e\u003cem\u003e\u003cspan data-contrast=\"none\"\u003eFSH\u003c/span\u003e\u003c/em\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;Follicle stimulating hormone,\u0026nbsp;\u003c/span\u003e\u003cem\u003e\u003cspan data-contrast=\"none\"\u003eLH\u0026nbsp;\u003c/span\u003e\u003c/em\u003e\u003cspan data-contrast=\"none\"\u003eLuteinizing hormone,\u0026nbsp;\u003c/span\u003e\u003cem\u003e\u003cspan data-contrast=\"none\"\u003eE\u003c/span\u003e\u003c/em\u003e\u003cem\u003e\u003cspan data-contrast=\"none\"\u003e\u003cspan data-fontsize=\"10.5\"\u003e2\u003c/span\u003e\u003c/span\u003e\u003c/em\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;Estradiol,\u0026nbsp;\u003c/span\u003e\u003cem\u003e\u003cspan data-contrast=\"none\"\u003eP\u003c/span\u003e\u003c/em\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;Progesterone,\u0026nbsp;\u003c/span\u003e\u003cem\u003e\u003cspan data-contrast=\"none\"\u003eTT\u0026nbsp;\u003c/span\u003e\u003c/em\u003e\u003cspan data-contrast=\"none\"\u003eTotal\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003etestosterone,\u0026nbsp;\u003c/span\u003e\u003cem\u003e\u003cspan data-contrast=\"none\"\u003eAMH\u003c/span\u003e\u003c/em\u003e\u003cspan data-contrast=\"none\"\u003eAnti-M\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026uuml;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003ellerian hormone,\u003c/span\u003e\u003cem\u003e\u003cspan data-contrast=\"none\"\u003ePRL\u003c/span\u003e\u003c/em\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;Prolactin. The rate of abnormal menstrual cycle was compared with Chi-square test.\u0026nbsp;\u003c/span\u003e\u003cem\u003e\u003cspan data-contrast=\"none\"\u003eP\u003c/span\u003e\u003c/em\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;\u0026lt; 0.05 was considered as statistically significant.\u003c/span\u003e\u003c/p\u003e\n\u003cp\u003e\u003cspan data-contrast=\"none\"\u003e\u003cspan data-fontsize=\"10.5\"\u003ea\u0026nbsp;\u003c/span\u003e\u003c/span\u003e\u003cem\u003e\u003cspan data-contrast=\"none\"\u003eP\u003c/span\u003e\u003c/em\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;\u0026lt; 0.05, group A\u0026nbsp;\u003c/span\u003e\u003cem\u003e\u003cspan data-contrast=\"none\"\u003evs.\u003c/span\u003e\u003c/em\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;group B\u003c/span\u003e\u003c/p\u003e\n\u003cp\u003e\u003cspan data-contrast=\"none\"\u003e\u003cspan data-fontsize=\"10.5\"\u003eb\u003c/span\u003e\u003c/span\u003e\u003cem\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;P\u003c/span\u003e\u003c/em\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;\u0026lt; 0.05, group A\u0026nbsp;\u003c/span\u003e\u003cem\u003e\u003cspan data-contrast=\"none\"\u003evs.\u003c/span\u003e\u003c/em\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;group C\u003c/span\u003e\u003c/p\u003e\n\u003cp\u003e\u003cspan data-contrast=\"none\"\u003e\u003cspan data-fontsize=\"10.5\"\u003ec\u003c/span\u003e\u003c/span\u003e\u003cem\u003e\u003cspan data-contrast=\"none\"\u003eP\u003c/span\u003e\u003c/em\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;\u0026lt; 0.05, group B\u0026nbsp;\u003c/span\u003e\u003cem\u003e\u003cspan data-contrast=\"none\"\u003evs.\u003c/span\u003e\u003c/em\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;group C\u003c/span\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cspan data-contrast=\"none\"\u003eDecline of oocyte quality and IVF outcome with increased age\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cspan data-contrast=\"none\"\u003eIn total t\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003ehirty-one cycles were canceled. The main reasons included\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003efollicular growth failure (10 cycles), failed oocyte retrieval at the time of follicle aspiration (6 cycles),\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003eno transplantable embryos (5 cycles\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e),\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003ea\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003ec\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003ecumulation\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;of embryos (5 cycles), and progesterone \u0026gt; 2.5 ng/ml on the trigger day (5 cycles). Compared with group C, groups A and B had significantly more\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e2PN oocytes,\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003etransplantable embryos, and higher rates of implantation and clinical pregnancy\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;(\u003c/span\u003e\u003cem\u003e\u003cspan data-contrast=\"none\"\u003eP\u003c/span\u003e\u003c/em\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;\u0026lt; 0.05).\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003eThe dosage of\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003erFSH\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e, duration of COS,\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003eendometrial thickness and E\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u003cspan data-fontsize=\"10.5\"\u003e2\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;levels on the trigger day, the number of retrieved oocytes,\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003eMⅡ\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;oocytes and embryos per ET, and the rates of miscarriage and multiple pregnancy did not significantly differ\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;among the three groups (\u003c/span\u003e\u003cem\u003e\u003cspan data-contrast=\"none\"\u003eP\u003c/span\u003e\u003c/em\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;\u0026gt; 0.05)\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;(Table 3).\u003c/span\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cspan data-contrast=\"none\"\u003eTable 3 COS, IVF outcomes and biochemical markers in FF\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"1\" data-table=\"\" data-tablelook=\"1184\"\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd data-celllook=\"257\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd data-celllook=\"257\"\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cspan data-contrast=\"none\"\u003egroup A\u0026nbsp;\u003c/span\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cspan data-contrast=\"none\"\u003e(n = 63)\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd data-celllook=\"257\"\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cspan data-contrast=\"none\"\u003eGroup B (n = 65)\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd data-celllook=\"273\"\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cspan data-contrast=\"none\"\u003eGroup C (n = 68)\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd data-celllook=\"1\"\u003e\n\u003cp\u003e\u003cspan data-contrast=\"none\"\u003eCOS\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd data-celllook=\"1\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd data-celllook=\"1\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd data-celllook=\"69649\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd data-celllook=\"0\"\u003e\n\u003cp\u003e\u003cspan data-contrast=\"none\"\u003eDosage of\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003erFSH\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;(IU)\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd data-celllook=\"0\"\u003e\n\u003cp\u003e\u003cspan data-contrast=\"none\"\u003e2476.31\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026plusmn;\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e379.34\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd data-celllook=\"0\"\u003e\n\u003cp\u003e\u003cspan data-contrast=\"none\"\u003e2556.69\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026plusmn;\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e419.57\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd data-celllook=\"0\"\u003e\n\u003cp\u003e\u003cspan data-contrast=\"none\"\u003e2586.90\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026plusmn;\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e587.63\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd data-celllook=\"0\"\u003e\n\u003cp\u003e\u003cspan data-contrast=\"none\"\u003eDuration of COS (d)\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd data-celllook=\"0\"\u003e\n\u003cp\u003e\u003cspan data-contrast=\"none\"\u003e9.89 \u0026plusmn; 2.85\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd data-celllook=\"0\"\u003e\n\u003cp\u003e\u003cspan data-contrast=\"none\"\u003e9.68\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026plusmn;\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e2.22\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd data-celllook=\"0\"\u003e\n\u003cp\u003e\u003cspan data-contrast=\"none\"\u003e9.33\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026plusmn;\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e2.34\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd data-celllook=\"0\"\u003e\n\u003cp\u003e\u003cspan data-contrast=\"none\"\u003eE\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u003cspan data-fontsize=\"10.5\"\u003e2\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;on trigger day (\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003epg\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e/mL)\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd data-celllook=\"0\"\u003e\n\u003cp\u003e\u003cspan data-contrast=\"none\"\u003e756.95\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026plusmn;\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e191.65\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd data-celllook=\"0\"\u003e\n\u003cp\u003e\u003cspan data-contrast=\"none\"\u003e740.23\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026plusmn;\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e180.47\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd data-celllook=\"0\"\u003e\n\u003cp\u003e\u003cspan data-contrast=\"none\"\u003e721.03\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026plusmn;\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e151.62\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd data-celllook=\"0\"\u003e\n\u003cp\u003e\u003cspan data-contrast=\"none\"\u003eEndometrial thickness (mm)\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd data-celllook=\"0\"\u003e\n\u003cp\u003e\u003cspan data-contrast=\"none\"\u003e9.\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e10 \u0026plusmn;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;1.89\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd data-celllook=\"0\"\u003e\n\u003cp\u003e\u003cspan data-contrast=\"none\"\u003e8.95 \u0026plusmn; 1.91\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd data-celllook=\"0\"\u003e\n\u003cp\u003e\u003cspan data-contrast=\"none\"\u003e8.81 \u0026plusmn; 1.61\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd data-celllook=\"0\"\u003e\n\u003cp\u003e\u003cspan data-contrast=\"none\"\u003eN\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003eumber of\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;oocytes retrieved\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd data-celllook=\"0\"\u003e\n\u003cp\u003e\u003cspan data-contrast=\"none\"\u003e2.98\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026plusmn;\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e0.67\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd data-celllook=\"0\"\u003e\n\u003cp\u003e\u003cspan data-contrast=\"none\"\u003e2.82\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026plusmn;\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e0.87\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd data-celllook=\"0\"\u003e\n\u003cp\u003e\u003cspan data-contrast=\"none\"\u003e2.68\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026plusmn;\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e0.90\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd data-celllook=\"0\"\u003e\n\u003cp\u003e\u003cspan data-contrast=\"none\"\u003eN\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003eumber of\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003eMⅡ\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd data-celllook=\"0\"\u003e\n\u003cp\u003e\u003cspan data-contrast=\"none\"\u003e2.54 \u0026plusmn; 0.94\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd data-celllook=\"0\"\u003e\n\u003cp\u003e\u003cspan data-contrast=\"none\"\u003e2.43 \u0026plusmn; 0.79\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd data-celllook=\"0\"\u003e\n\u003cp\u003e\u003cspan data-contrast=\"none\"\u003e2.37 \u0026plusmn; 0.74\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd data-celllook=\"0\"\u003e\n\u003cp\u003e\u003cspan data-contrast=\"none\"\u003eN\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003eumber of\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;2PN\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u003cspan data-fontsize=\"10.5\"\u003eab\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd data-celllook=\"0\"\u003e\n\u003cp\u003e\u003cspan data-contrast=\"none\"\u003e2.08 \u0026plusmn; 0.93\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd data-celllook=\"0\"\u003e\n\u003cp\u003e\u003cspan data-contrast=\"none\"\u003e1.87 \u0026plusmn; 0.87\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd data-celllook=\"65536\"\u003e\n\u003cp\u003e\u003cspan data-contrast=\"none\"\u003e1.36 \u0026plusmn; 0.58\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd data-celllook=\"0\"\u003e\n\u003cp\u003e\u003cspan data-contrast=\"none\"\u003eN\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003eumber of\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;transplantable embryos\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u003cspan data-fontsize=\"10.5\"\u003eab\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd data-celllook=\"0\"\u003e\n\u003cp\u003e\u003cspan data-contrast=\"none\"\u003e2.02 \u0026plusmn; 0.57\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd data-celllook=\"0\"\u003e\n\u003cp\u003e\u003cspan data-contrast=\"none\"\u003e1.85 \u0026plusmn; 0.71\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd data-celllook=\"0\"\u003e\n\u003cp\u003e\u003cspan data-contrast=\"none\"\u003e1.22 \u0026plusmn; 0.49\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd data-celllook=\"0\"\u003e\n\u003cp\u003e\u003cspan data-contrast=\"none\"\u003eCancel cycle rate (%)\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd data-celllook=\"0\"\u003e\n\u003cp\u003e\u003cspan data-contrast=\"none\"\u003e11.11 % (7/63)\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd data-celllook=\"0\"\u003e\n\u003cp\u003e\u003cspan data-contrast=\"none\"\u003e12.31% (8/65)\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd data-celllook=\"69904\"\u003e\n\u003cp\u003e\u003cspan data-contrast=\"none\"\u003e23.53% (16/68)\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd data-celllook=\"0\"\u003e\n\u003cp\u003e\u003cspan data-contrast=\"none\"\u003eET\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd data-celllook=\"0\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd data-celllook=\"0\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd data-celllook=\"69904\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd data-celllook=\"0\"\u003e\n\u003cp\u003e\u003cspan data-contrast=\"none\"\u003eN\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003eumber of e\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003embryos per ET\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd data-celllook=\"0\"\u003e\n\u003cp\u003e\u003cspan data-contrast=\"none\"\u003e1.43 \u0026plusmn; 0.50\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd data-celllook=\"0\"\u003e\n\u003cp\u003e\u003cspan data-contrast=\"none\"\u003e1.44 \u0026plusmn; 0.50\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd data-celllook=\"0\"\u003e\n\u003cp\u003e\u003cspan data-contrast=\"none\"\u003e1.33 \u0026plusmn; 0.47 \u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd data-celllook=\"0\"\u003e\n\u003cp\u003e\u003cspan data-contrast=\"none\"\u003eImplantation rate (%)\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u003cspan data-fontsize=\"10.5\"\u003eab\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd data-celllook=\"0\"\u003e\n\u003cp\u003e\u003cspan data-contrast=\"none\"\u003e30.00% (24/80)\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd data-celllook=\"0\"\u003e\n\u003cp\u003e\u003cspan data-contrast=\"none\"\u003e24.39% (20/82)\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd data-celllook=\"0\"\u003e\n\u003cp\u003e\u003cspan data-contrast=\"none\"\u003e10.14% (7/69)\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd data-celllook=\"0\"\u003e\n\u003cp\u003e\u003cspan data-contrast=\"none\"\u003eClinical pregnancy rate (%)\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u003cspan data-fontsize=\"10.5\"\u003eab\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd data-celllook=\"0\"\u003e\n\u003cp\u003e\u003cspan data-contrast=\"none\"\u003e37.50% (21/56)\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd data-celllook=\"0\"\u003e\n\u003cp\u003e\u003cspan data-contrast=\"none\"\u003e29.82% (17/57)\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd data-celllook=\"0\"\u003e\n\u003cp\u003e\u003cspan data-contrast=\"none\"\u003e11.54% (6/52)\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd data-celllook=\"0\"\u003e\n\u003cp\u003e\u003cspan data-contrast=\"none\"\u003eMiscarriage rate (%)\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd data-celllook=\"0\"\u003e\n\u003cp\u003e\u003cspan data-contrast=\"none\"\u003e9.52% (2/21)\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd data-celllook=\"0\"\u003e\n\u003cp\u003e\u003cspan data-contrast=\"none\"\u003e23.53% (4/17)\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd data-celllook=\"0\"\u003e\n\u003cp\u003e\u003cspan data-contrast=\"none\"\u003e33.33% (2/6)\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd data-celllook=\"0\"\u003e\n\u003cp\u003e\u003cspan data-contrast=\"none\"\u003eMultiple pregnancy rate (%)\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd data-celllook=\"0\"\u003e\n\u003cp\u003e\u003cspan data-contrast=\"none\"\u003e14.29% (3/21)\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd data-celllook=\"0\"\u003e\n\u003cp\u003e\u003cspan data-contrast=\"none\"\u003e17.65% (3/17)\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd data-celllook=\"0\"\u003e\n\u003cp\u003e\u003cspan data-contrast=\"none\"\u003e16.67% (1/6)\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd data-celllook=\"0\"\u003e\n\u003cp\u003e\u003cspan data-contrast=\"none\"\u003eIn FF\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd data-celllook=\"0\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd data-celllook=\"0\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd data-celllook=\"69649\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd data-celllook=\"0\"\u003e\n\u003cp\u003e\u003cem\u003e\u003cspan data-contrast=\"none\"\u003eGDF9\u003c/span\u003e\u003c/em\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;(\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003epg\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e/ml)\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u003cspan data-fontsize=\"10.5\"\u003eabc\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd data-celllook=\"0\"\u003e\n\u003cp\u003e\u003cspan data-contrast=\"none\"\u003e1020 \u0026plusmn; 117\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd data-celllook=\"0\"\u003e\n\u003cp\u003e\u003cspan data-contrast=\"none\"\u003e904\u0026nbsp;\u003c/span\u003e\u003cstrong\u003e\u003cspan data-contrast=\"none\"\u003e\u0026plusmn;\u0026nbsp;\u003c/span\u003e\u003c/strong\u003e\u003cspan data-contrast=\"none\"\u003e70\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd data-celllook=\"0\"\u003e\n\u003cp\u003e\u003cspan data-contrast=\"none\"\u003e671\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026plusmn; 53\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd data-celllook=\"256\"\u003e\n\u003cp\u003e\u003cem\u003e\u003cspan data-contrast=\"none\"\u003eBMP15\u003c/span\u003e\u003c/em\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;(\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003epg\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e/ml)\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u003cspan data-fontsize=\"10.5\"\u003eabc\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd data-celllook=\"256\"\u003e\n\u003cp\u003e\u003cspan data-contrast=\"none\"\u003e666 \u0026plusmn; 40\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd data-celllook=\"256\"\u003e\n\u003cp\u003e\u003cspan data-contrast=\"none\"\u003e643\u0026nbsp;\u003c/span\u003e\u003cstrong\u003e\u003cspan data-contrast=\"none\"\u003e\u0026plusmn;\u0026nbsp;\u003c/span\u003e\u003c/strong\u003e\u003cspan data-contrast=\"none\"\u003e46\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd data-celllook=\"256\"\u003e\n\u003cp\u003e\u003cspan data-contrast=\"none\"\u003e420 \u0026plusmn; 40\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cspan data-contrast=\"none\"\u003eData are presented as mean \u0026plusmn; SD or percentage (number).\u0026nbsp;\u003c/span\u003e\u003cstrong\u003e\u003cem\u003e\u003cspan data-contrast=\"none\"\u003eAbbreviations:\u0026nbsp;\u003c/span\u003e\u003c/em\u003e\u003c/strong\u003e\u003cem\u003e\u003cspan data-contrast=\"none\"\u003eCOS\u003c/span\u003e\u003c/em\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;Controlled ovarian stimulation,\u0026nbsp;\u003c/span\u003e\u003cem\u003e\u003cspan data-contrast=\"none\"\u003eE\u003c/span\u003e\u003c/em\u003e\u003cem\u003e\u003cspan data-contrast=\"none\"\u003e\u003cspan data-fontsize=\"10.5\"\u003e2\u003c/span\u003e\u003c/span\u003e\u003c/em\u003e\u003cspan data-contrast=\"none\"\u003eEstradiol,\u0026nbsp;\u003c/span\u003e\u003cem\u003e\u003cspan data-contrast=\"none\"\u003eET\u003c/span\u003e\u003c/em\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;Embryos transferred,\u0026nbsp;\u003c/span\u003e\u003cem\u003e\u003cspan data-contrast=\"none\"\u003eFF\u0026nbsp;\u003c/span\u003e\u003c/em\u003e\u003cspan data-contrast=\"none\"\u003eFollicle fluid,\u0026nbsp;\u003c/span\u003e\u003cem\u003e\u003cspan data-contrast=\"none\"\u003eGDF9\u003c/span\u003e\u003c/em\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;Growth differentiation factor 9,\u0026nbsp;\u003c/span\u003e\u003cem\u003e\u003cspan data-contrast=\"none\"\u003eBMP15\u003c/span\u003e\u003c/em\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;Bone morphogenetic protein-15.\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003eChi-squared test was used to compare the rates of\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003ematuration\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e,\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003efertilization, cleaved embryo, higher quality embryo, cancel cycle, implantation, clinical pregnancy, miscarriage and multiple pregnancy between\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003ethe\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003etwo groups.\u003c/span\u003e\u003cem\u003e\u003cspan data-contrast=\"none\"\u003eP\u003c/span\u003e\u003c/em\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;\u0026lt; 0.05 was considered as statistically significant.\u003c/span\u003e\u003c/p\u003e\n\u003cp\u003e\u003cspan data-contrast=\"none\"\u003e\u003cspan data-fontsize=\"10.5\"\u003ea\u0026nbsp;\u003c/span\u003e\u003c/span\u003e\u003cem\u003e\u003cspan data-contrast=\"none\"\u003eP\u003c/span\u003e\u003c/em\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;\u0026lt; 0.05, group A\u0026nbsp;\u003c/span\u003e\u003cem\u003e\u003cspan data-contrast=\"none\"\u003evs.\u003c/span\u003e\u003c/em\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;group C\u003c/span\u003e\u003c/p\u003e\n\u003cp\u003e\u003cspan data-contrast=\"none\"\u003e\u003cspan data-fontsize=\"10.5\"\u003eb\u003c/span\u003e\u003c/span\u003e\u003cem\u003e\u003cspan data-contrast=\"none\"\u003eP\u003c/span\u003e\u003c/em\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;\u0026lt; 0.05, group B\u0026nbsp;\u003c/span\u003e\u003cem\u003e\u003cspan data-contrast=\"none\"\u003evs.\u003c/span\u003e\u003c/em\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;group C\u003c/span\u003e\u003c/p\u003e\n\u003cp\u003e\u003cspan data-contrast=\"none\"\u003e\u003cspan data-fontsize=\"10.5\"\u003ec\u003c/span\u003e\u003c/span\u003e\u003cem\u003e\u003cspan data-contrast=\"none\"\u003eP\u003c/span\u003e\u003c/em\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;\u0026lt; 0.05, group A\u0026nbsp;\u003c/span\u003e\u003cem\u003e\u003cspan data-contrast=\"none\"\u003evs.\u003c/span\u003e\u003c/em\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;group B\u003c/span\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cspan data-contrast=\"none\"\u003eDecreased expression of\u0026nbsp;\u003c/span\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cspan data-contrast=\"none\"\u003eGDF9 and BMP15\u003c/span\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;in FF with increased age\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cspan data-contrast=\"none\"\u003eAs shown in Table 3, the concentration of\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003eGDF9\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;(\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e101\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e2\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;\u0026plusmn;\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e117\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003ep\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003eg/ml\u003c/span\u003e\u003cem\u003e\u003cspan data-contrast=\"none\"\u003evs.\u003c/span\u003e\u003c/em\u003e\u003cspan data-contrast=\"none\"\u003e90\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e4\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026plusmn;\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e70\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;p\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003eg/ml\u003c/span\u003e\u003cem\u003e\u003cspan data-contrast=\"none\"\u003evs.\u003c/span\u003e\u003c/em\u003e\u003cspan data-contrast=\"none\"\u003e671\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026plusmn;\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e5\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e3\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003ep\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003eg/ml\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e) and\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003eBMP15\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;(\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e666\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;\u0026plusmn;\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e40\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;p\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003eg/ml\u0026nbsp;\u003c/span\u003e\u003cem\u003e\u003cspan data-contrast=\"none\"\u003evs.\u003c/span\u003e\u003c/em\u003e\u003cspan data-contrast=\"none\"\u003e64\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e3\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;\u0026plusmn;\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e4\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e6\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;p\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003eg/ml\u0026nbsp;\u003c/span\u003e\u003cem\u003e\u003cspan data-contrast=\"none\"\u003evs.\u003c/span\u003e\u003c/em\u003e\u003cspan data-contrast=\"none\"\u003e4\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e20\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026plusmn;\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e40\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003ep\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003eg/ml\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e) in FF have differed significantly between the three groups (\u003c/span\u003e\u003cem\u003e\u003cspan data-contrast=\"none\"\u003eP\u003c/span\u003e\u003c/em\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;\u0026lt; 0.05), with an obvious trend of decline with increased age.\u003c/span\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cspan data-contrast=\"none\"\u003eDecreased expression of\u0026nbsp;\u003c/span\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cspan data-contrast=\"none\"\u003eGDF9 and BMP15 in\u003c/span\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;GCs with increased age\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cspan data-contrast=\"none\"\u003eOnly GCs in FF with oocyte were detected for mRNA and protein. Therefore, there were 61, 60 and 59 samples\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003efrom\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;group A, group B and group C, respectively. The CT values of\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003ehousekeeping genes did not differ significantly among the three groups (\u003c/span\u003e\u003cem\u003e\u003cspan data-contrast=\"none\"\u003eP\u003c/span\u003e\u003c/em\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;\u0026gt; 0.05). As shown in Figures 1A and\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e1\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003eB, the expression of\u0026nbsp;\u003c/span\u003e\u003cem\u003e\u003cspan data-contrast=\"none\"\u003eGDF9\u003c/span\u003e\u003c/em\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;and\u0026nbsp;\u003c/span\u003e\u003cem\u003e\u003cspan data-contrast=\"none\"\u003eBMP15\u0026nbsp;\u003c/span\u003e\u003c/em\u003e\u003cspan data-contrast=\"none\"\u003ein GCs\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e, at the levels of both\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003emRNA and protein, have differed significantly\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003ebetween the three groups (\u003c/span\u003e\u003cem\u003e\u003cspan data-contrast=\"none\"\u003eP\u0026nbsp;\u003c/span\u003e\u003c/em\u003e\u003cspan data-contrast=\"none\"\u003e\u0026lt; 0.05)\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e. Both showed a trend of decline with increased age. As shown in\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003eFigure\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;1C, the\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003esingle band at 51\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003ekDa\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;represented\u0026nbsp;\u003c/span\u003e\u003cem\u003e\u003cspan data-contrast=\"none\"\u003eGDF9\u003c/span\u003e\u003c/em\u003e\u003cspan data-contrast=\"none\"\u003e, while the single band at 45\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003ekDa\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;represented\u0026nbsp;\u003c/span\u003e\u003cem\u003e\u003cspan data-contrast=\"none\"\u003eBMP15\u003c/span\u003e\u003c/em\u003e\u003cspan data-contrast=\"none\"\u003e.\u003c/span\u003e\u003c/p\u003e\n\u003cp\u003e\u003cspan data-contrast=\"none\"\u003eAs shown in Table 4,\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003et\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003ehe relative protein levels in GCs\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003ew\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003eas\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;positively correlated with the relative mRNA levels in GCs and protein concentration\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003es\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;in FF\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003eby\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;Pearson\u0026rsquo;s correlation\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;analysis\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;(\u003c/span\u003e\u003cem\u003e\u003cspan data-contrast=\"none\"\u003eP\u003c/span\u003e\u003c/em\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;\u0026lt; 0.05).\u003c/span\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cspan data-contrast=\"none\"\u003eTable 4 Correlation between the proteins and mRNA levels\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"1\" data-table=\"\" data-tablelook=\"1184\"\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd colspan=\"1\" rowspan=\"2\" data-celllook=\"4369\"\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cspan data-contrast=\"none\"\u003erelative protein levels\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cspan data-contrast=\"none\"\u003ein GCs\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" data-celllook=\"4369\"\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;relative mRNA levels in GCs\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" data-celllook=\"4369\"\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cspan data-contrast=\"none\"\u003eprotein concentration in FF\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd data-celllook=\"4369\"\u003e\n\u003cp\u003e\u003cem\u003e\u003cspan data-contrast=\"none\"\u003er\u003c/span\u003e\u003c/em\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd data-celllook=\"4369\"\u003e\n\u003cp\u003e\u003cem\u003e\u003cspan data-contrast=\"none\"\u003eP\u003c/span\u003e\u003c/em\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd data-celllook=\"4369\"\u003e\n\u003cp\u003e\u003cem\u003e\u003cspan data-contrast=\"none\"\u003er\u003c/span\u003e\u003c/em\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd data-celllook=\"4369\"\u003e\n\u003cp\u003e\u003cem\u003e\u003cspan data-contrast=\"none\"\u003eP\u003c/span\u003e\u003c/em\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd data-celllook=\"4369\"\u003e\n\u003cp\u003e\u003cspan data-contrast=\"none\"\u003eGDF9\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd data-celllook=\"4369\"\u003e\n\u003cp\u003e\u003cspan data-contrast=\"none\"\u003e0.704\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd data-celllook=\"4369\"\u003e\n\u003cp\u003e\u003cspan data-contrast=\"none\"\u003e\u0026lt; 0.001\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd data-celllook=\"4369\"\u003e\n\u003cp\u003e\u003cspan data-contrast=\"none\"\u003e0.501\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd data-celllook=\"4369\"\u003e\n\u003cp\u003e\u003cspan data-contrast=\"none\"\u003e0.001\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd data-celllook=\"4369\"\u003e\n\u003cp\u003e\u003cspan data-contrast=\"none\"\u003eBMP15\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd data-celllook=\"4369\"\u003e\n\u003cp\u003e\u003cspan data-contrast=\"none\"\u003e0.737\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd data-celllook=\"4369\"\u003e\n\u003cp\u003e\u003cspan data-contrast=\"none\"\u003e\u0026lt; 0.001\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd data-celllook=\"4369\"\u003e\n\u003cp\u003e\u003cspan data-contrast=\"none\"\u003e0.701\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd data-celllook=\"4369\"\u003e\n\u003cp\u003e\u003cspan data-contrast=\"none\"\u003e\u0026lt; 0.001\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cspan data-contrast=\"none\"\u003eThe ratio of GDF9/BMP15 mRNA levels\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cspan data-contrast=\"none\"\u003eThe ratio of GDF9/BMP15 mRNA levels\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003eof\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003ethe\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003ethree groups\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003ewere\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;compared using one-way ANOVA. The ratio of GDF9/BMP15 mRNA levels\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003efor groups A, B and C\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003ewere 1.23 (1.00-1.35), 1.23 (1.08-1.38), and 1.20 (1.09-1.33), respectively.\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;N\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003eo significant difference\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003ewas detected between\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;the groups (\u003c/span\u003e\u003cem\u003e\u003cspan data-contrast=\"none\"\u003eP\u003c/span\u003e\u003c/em\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;\u0026gt; 0.05). Linear regression equation for the mRNA levels of GDF9 against BMP15 in e\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003each\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;sample was established\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;as\u003c/span\u003e\u003cem\u003e\u003cspan data-contrast=\"none\"\u003ey\u003c/span\u003e\u003c/em\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;= 1.097\u003c/span\u003e\u003cem\u003e\u003cspan data-contrast=\"none\"\u003ex\u003c/span\u003e\u003c/em\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;+ 0.047.\u003c/span\u003e\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003e\u003cspan data-contrast=\"none\"\u003eWe have proven t\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003ehrough this study\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003ethat the oocyte quality and IVF outcome will decrease with increased age among patients with POR, in particular those over 40, which also correlated with decline of\u0026nbsp;\u003c/span\u003e\u003cem\u003e\u003cspan data-contrast=\"none\"\u003eGDF9\u003c/span\u003e\u003c/em\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;and\u0026nbsp;\u003c/span\u003e\u003cem\u003e\u003cspan data-contrast=\"none\"\u003eBMP15\u003c/span\u003e\u003c/em\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;expression in FF and GCs.\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003eThe ratio of GDF9/BMP15 mRNA levels\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003ehas remained to be relatively\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;constant, and the protein and mRNA levels were positively correlated\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003ein\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003eboth\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003eFF and GCs.\u003c/span\u003e\u003c/p\u003e\n\u003cp\u003e\u003cspan data-contrast=\"none\"\u003eAlthough the quantity\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003eof\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003eoocyte\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003ewas\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003epoor\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;in all groups in this study, the numbers of normal fertilized oocytes and transplantable embryos have decreased\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003esteeply\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;to 1.36 and 1.22 in poor responders over 40\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e, respectively\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e. And the oocyte quality is one of the most critical factors for pregnancy. Similar to previous reports, the rates of implantation and clinical pregnancy in elder patients (\u0026gt; 40) have decreased to 10.14% and 11.54%, respectively\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e[7,8]\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e. This seems to have reflected the poor quantity and quality of the oocytes from elder poor responders whom may benefit less from IVF treatment. For poor responders between 35 and 40, the rates of implantation and clinical pregnancy were 24.39% and 29.82%, respectively\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e. B\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003eoth\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003ewere\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003elower than those of under 35\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e,\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;albeit with no statistical significance.\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003eResearchers have p\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003erevious\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003ely\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;proposed that 35\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e-\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e37 year of age to be the threshold for decreased rate of euploidy embryos and live births\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e[25]\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e. Such patients should therefore consider IVF treatment earlier. Meanwhile, younger poor responders (\u0026lt; 35) still have reasonable number of transplantable embryos (2.02\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026plusmn;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;0.57) and pregnancy rate (37.50%), and should be actively treated. Of note, the miscarriage rate may also rise with increased age\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e[8]\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e. The underlying mechanisms for age-related decline of oocyte quality and IVF outcome are variable, including\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003echromosomal aberration in oocytes and embryos due to advanced maternal age\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e[26-28]\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e. The autocrine function of oocytes in POR is also important but not yet fully understood.\u003c/span\u003e\u003c/p\u003e\n\u003cp\u003e\u003cspan data-contrast=\"none\"\u003eGDF9 and BMP15 secreted by the oocyte play an important role in the cross-talk between cumulus GCs and the oocytes\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e[22]\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e.\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;The functions of GDF9 and BMP15\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003ein\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;follicular development include suppressing GCs apoptosis and promoting cell proliferation\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e[29]\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e, enhancing the effect of FSH and insulin-like growth factor-\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003eI\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;(IGF- I) on GCs, then providing more E\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u003cspan data-fontsize=\"10.5\"\u003e2\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;and glycolysis for oocyte\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e[30,31]\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e. GDF9 and BMP15 can also prevent premature luteinization and promote normal expansion of cumulus cells till the LH surge\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e[32,33]\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e.\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003eIn turn, GCs are necessary to nourish the oocytes and promote their maturation\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e[17,34]\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e. Therefore, altered expression and/or function of GDF9 and BMP15 may result in abnormal\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003efolliculogenesis\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;and poor oocyte quality.\u003c/span\u003e\u003c/p\u003e\n\u003cp\u003e\u003cspan data-contrast=\"none\"\u003eDeclined\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;expression of the\u0026nbsp;\u003c/span\u003e\u003cem\u003e\u003cspan data-contrast=\"none\"\u003eGDF9\u003c/span\u003e\u003c/em\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;and\u0026nbsp;\u003c/span\u003e\u003cem\u003e\u003cspan data-contrast=\"none\"\u003eBMP15\u003c/span\u003e\u003c/em\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;genes has previously been discovered among patients with DOR\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e[21,35]\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e. In this study, we further proved that such de\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003ecline\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;is associated with increased age, particularly for those over 40. FF and GCs\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003eform\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;the microenvironment of the oocytes, and certain components of them may reflect the metabolism and endocrine status of the oocyte\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e[34,36]\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e. In this study,\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003ewe\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003efound\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003ethat\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003ethe\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003erelative protein levels in GCs\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003ehave\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003ecorrelate\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003ed\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;with the relative mRNA levels in GCs and protein concentration in FF. This\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;has\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;reflected the si\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003emilar\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;changes of the two proteins in FF and GCs\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e,\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;both\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003eof which\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003ecan reflect the status of oocyte. Furthermore,\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003ethe decline of\u0026nbsp;\u003c/span\u003e\u003cem\u003e\u003cspan data-contrast=\"none\"\u003eGDF9\u003c/span\u003e\u003c/em\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;and\u0026nbsp;\u003c/span\u003e\u003cem\u003e\u003cspan data-contrast=\"none\"\u003eBMP15\u003c/span\u003e\u003c/em\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;expression was accompanied with poor oocyte quality and IVF outcome in elder patients. Compared with group A (\u0026lt; 35), group B (35\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e-\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e40) had significantly lower expression. The\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003eir\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;oocyte quality and IVF outcome were also poorer albeit with no statistical significance. Hence,\u0026nbsp;\u003c/span\u003e\u003cem\u003e\u003cspan data-contrast=\"none\"\u003eGDF9\u003c/span\u003e\u003c/em\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;and\u0026nbsp;\u003c/span\u003e\u003cem\u003e\u003cspan data-contrast=\"none\"\u003eBMP15\u003c/span\u003e\u003c/em\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;may provide more sensitive and earlier biomarkers for the fertility of poor ovarian responders. Declined expression of the two may, at least in part, account for the age-related poor oocyte quality in poor responders.\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;Physiologically,\u0026nbsp;\u003c/span\u003e\u003cem\u003e\u003cspan data-contrast=\"none\"\u003eGDF9\u003c/span\u003e\u003c/em\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;and\u0026nbsp;\u003c/span\u003e\u003cem\u003e\u003cspan data-contrast=\"none\"\u003eBMP15\u003c/span\u003e\u003c/em\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;can promote the expansion of cumulus GCs till the LH surge\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e[32,33]\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e. For elder poor responders, the steep dec\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003eline\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;in\u0026nbsp;\u003c/span\u003e\u003cem\u003e\u003cspan data-contrast=\"none\"\u003eGDF9\u003c/span\u003e\u003c/em\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;and\u0026nbsp;\u003c/span\u003e\u003cem\u003e\u003cspan data-contrast=\"none\"\u003eBMP15\u0026nbsp;\u003c/span\u003e\u003c/em\u003e\u003cspan data-contrast=\"none\"\u003eexpression may affect the response of GCs to LH surge. For such patients,\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003eexogenous LH in\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003econjunct with\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003erFSH\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;may compensate the insufficient function of LH and improve the outcome of IVF-ET treatment\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e[37]\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e.\u003c/span\u003e\u003c/p\u003e\n\u003cp\u003e\u003cspan data-contrast=\"none\"\u003eOf note\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e, the ratio of GDF9/BMP15 mRNA levels in GCs\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003ehas remained relatively constant\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003eamong\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;the three groups\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e, and\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003et\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003eherefore m\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003eay\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;not\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003ebe\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003eassoci\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003eate\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003ed\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;with the onset of early DOR. Crawford et al\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e[38]\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;also\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003efound\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;the ratio of GDF9\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e/\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003eBMP15 mRNA\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003eto be\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003estrongly\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003ereserved in\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;each species but differed markedly between species. Oocyte derived from poly-ovulatory species exhibited higher GDF9\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e/\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003eBMP15 ratios,\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003ee.g.,\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003emouse (5.19) and rat (3.65),\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003ecompared with species with\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;low ovulation rates,\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003ee.g.,\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003esheep (1.26), cow (0.24) and deer (0.10)\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e[38]\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e.\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003eIn the present study, t\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003ehe ratio of human GCs (1.20-1.23) was similar\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003eto that of\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003elow o\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003evulatory species such as sheep (1.26). GDF9 and BMP15\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003ework\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;synergistic\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003eally\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;to influence\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003ethe proliferation of\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003eGCs, and GCs of each species\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003emight\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003ehave evolved to respond to unique GDF9\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e/\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003eBMP15 ratios\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e[39]\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e.\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;Nevertheless\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e, Crawford\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003eet al.\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;found the ratio in\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003epig\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003eoocyte\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003es\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;(poly-ovulatory species)\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003eto be\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003ealso\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003elow (0.51)\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e[38]\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e.\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003eTherefore, t\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003ehe ratio of GDF9\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e/\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003eBMP15\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003emay\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;not\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003ebe\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003ethe only factor which\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;can influence\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;the ovulation rate. The underlying mechanism of the GDF9\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e/\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003eBMP15\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003eratio\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003eand ovulation rate\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003edemands\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;further research.\u003c/span\u003e\u003c/p\u003e\n\u003cp\u003e\u003cspan data-contrast=\"none\"\u003eThe limitation of this study lies in its relatively small\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;sample size and lack\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;of\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;livebirth\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003erate\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;d\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003eue to the limited study period.\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;Moreover, as the main purpose of this study was to evaluate\u0026nbsp;\u003c/span\u003e\u003cem\u003e\u003cspan data-contrast=\"none\"\u003eGDF9\u003c/span\u003e\u003c/em\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;and\u0026nbsp;\u003c/span\u003e\u003cem\u003e\u003cspan data-contrast=\"none\"\u003eBMP15\u003c/span\u003e\u003c/em\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;expression in female age groups, we have excluded ICSI cycles due to male factors. The latter, in particular advanced paternal age, may also affect the embryo quality and outcome of IVF\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e-ET\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;treatment.\u003c/span\u003e\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003e\u003cspan lang=\"EN-US\" xml:lang=\"EN-US\" data-contrast=\"none\"\u003eIn summary, we have discovered an age-related decline\u0026nbsp;\u003c/span\u003e\u003cspan lang=\"EN-US\" xml:lang=\"EN-US\" data-contrast=\"none\"\u003ein\u003c/span\u003e\u003cspan lang=\"EN-US\" xml:lang=\"EN-US\" data-contrast=\"none\"\u003eGDF9\u003c/span\u003e\u003cspan lang=\"EN-US\" xml:lang=\"EN-US\" data-contrast=\"none\"\u003e\u0026nbsp;and\u0026nbsp;\u003c/span\u003e\u003cspan lang=\"EN-US\" xml:lang=\"EN-US\" data-contrast=\"none\"\u003eBMP15\u003c/span\u003e\u003cspan lang=\"EN-US\" xml:lang=\"EN-US\" data-contrast=\"none\"\u003e\u0026nbsp;expression among patients with POR, which may in part account for the age-related poor oocyte quality and IVF outcome.\u0026nbsp;\u003c/span\u003e\u003cspan lang=\"EN-US\" xml:lang=\"EN-US\" data-contrast=\"none\"\u003eIdentification of the heterogeneity may facilitate design of individualized protocols to\u0026nbsp;\u003c/span\u003e\u003cspan lang=\"EN-US\" xml:lang=\"EN-US\" data-contrast=\"none\"\u003eattain\u003c/span\u003e\u003cspan lang=\"EN-US\" xml:lang=\"EN-US\" data-contrast=\"none\"\u003e\u0026nbsp;better treatment outcome and avoid repeated cycles.\u003c/span\u003e\u003cspan lang=\"EN-US\" xml:lang=\"EN-US\" data-contrast=\"none\"\u003e\u0026nbsp;L\u003c/span\u003e\u003cspan lang=\"EN-US\" xml:lang=\"EN-US\" data-contrast=\"none\"\u003earger cohort\u003c/span\u003e\u003cspan lang=\"EN-US\" xml:lang=\"EN-US\" data-contrast=\"none\"\u003e\u0026nbsp;studies are\u003c/span\u003e\u003cspan lang=\"EN-US\" xml:lang=\"EN-US\" data-contrast=\"none\"\u003e\u0026nbsp;required to validate the result\u003c/span\u003e\u003cspan lang=\"EN-US\" xml:lang=\"EN-US\" data-contrast=\"none\"\u003es\u003c/span\u003e\u003cspan lang=\"EN-US\" xml:lang=\"EN-US\" data-contrast=\"none\"\u003e\u0026nbsp;of this study. Researchers have recently suggested\u0026nbsp;\u003c/span\u003e\u003cspan lang=\"EN-US\" xml:lang=\"EN-US\" data-contrast=\"none\"\u003ethat exogenous recombinant\u0026nbsp;\u003c/span\u003e\u003cspan lang=\"EN-US\" xml:lang=\"EN-US\" data-contrast=\"none\"\u003eGDF9\u003c/span\u003e\u003cspan lang=\"EN-US\" xml:lang=\"EN-US\" data-contrast=\"none\"\u003e\u0026nbsp;and/or\u0026nbsp;\u003c/span\u003e\u003cspan lang=\"EN-US\" xml:lang=\"EN-US\" data-contrast=\"none\"\u003eBMP15\u003c/span\u003e\u003cspan lang=\"EN-US\" xml:lang=\"EN-US\" data-contrast=\"none\"\u003ecan enhance\u003c/span\u003e\u003cspan lang=\"EN-US\" xml:lang=\"EN-US\" data-contrast=\"none\"\u003e\u0026nbsp;blastocyst formation during IVF treatment\u0026nbsp;\u003c/span\u003e\u003cspan lang=\"EN-US\" xml:lang=\"EN-US\" data-contrast=\"none\"\u003e[40,41]\u003c/span\u003e\u003cspan lang=\"EN-US\" xml:lang=\"EN-US\" data-contrast=\"none\"\u003e. Should such strategy be adapted for poor ovarian responders, dosage\u003c/span\u003e\u003cspan lang=\"EN-US\" xml:lang=\"EN-US\" data-contrast=\"none\"\u003es\u003c/span\u003e\u003cspan lang=\"EN-US\" xml:lang=\"EN-US\" data-contrast=\"none\"\u003e\u0026nbsp;dependent on age and basal levels of\u0026nbsp;\u003c/span\u003e\u003cspan lang=\"EN-US\" xml:lang=\"EN-US\" data-contrast=\"none\"\u003eGDF9\u003c/span\u003e\u003cspan lang=\"EN-US\" xml:lang=\"EN-US\" data-contrast=\"none\"\u003e\u0026nbsp;and\u0026nbsp;\u003c/span\u003e\u003cspan lang=\"EN-US\" xml:lang=\"EN-US\" data-contrast=\"none\"\u003eBMP15\u003c/span\u003e\u003cspan lang=\"EN-US\" xml:lang=\"EN-US\" data-contrast=\"none\"\u003e\u0026nbsp;expression\u0026nbsp;\u003c/span\u003e\u003cspan lang=\"EN-US\" xml:lang=\"EN-US\" data-contrast=\"none\"\u003emay\u003c/span\u003e\u003cspan lang=\"EN-US\" xml:lang=\"EN-US\" data-contrast=\"none\"\u003e\u0026nbsp;be considered.\u003c/span\u003e\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003e\u003cspan data-contrast=\"none\"\u003eAcknowledgement\u0026nbsp;\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cspan data-contrast=\"none\"\u003eWe thank our colleagues at the Reproductive Medicine Center of Sichuan Provincial Women\u0026rsquo;s and Children\u0026rsquo;s Hospital for their assistance in sample collection. We thank\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003eXiang\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003ey\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003eu\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;Li\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003eand\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003eShiQi\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;Chen\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003efor\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003etheir\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;assistance in\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003edata analysis. We\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003egreatly\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;appreciate all patients\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003efor their\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;participat\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003eion\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;in this study.\u003c/span\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cspan data-contrast=\"none\"\u003eAuthor\u0026rsquo;s contributions\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cspan data-contrast=\"none\"\u003eYan Gong designed the study and wrote the manuscript. Jesse Li-Ling contributed to revise the article. Hao Tan\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003edesigned the experiments.\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003eTaiqing\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;Zhong performed the experiments.\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003eJiajing\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;Wei and Dongsheng\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003eXiong\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;participated in sample collection. All authors\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003ehave\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003eread and approved the final manuscript.\u003c/span\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cspan data-contrast=\"none\"\u003eFunding\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cspan data-contrast=\"none\"\u003eThis stu\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003edy was\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003esponsored\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;by\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;the Scientific Research Pro\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003egram\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;of Sichuan Medical Association\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;(No.\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003eS17060\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e),\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003ethe Technology Innovation\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;Pro\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003egram\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;of\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003ethe\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003eScience and Technology Bureau of Chengdu\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;(No.\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e2018-YF05-00247-SN\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e),\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003et\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003ehe Scientific Research Project of Sichuan Provincial Health Commission (No. 20PJ123),\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003eScience and Technology Innovation Fund of Sichuan Provincial Hospital for Women and Children\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e(\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003eNo.\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e20180205) and the Innovation and Entrepreneurship Training Program for College Students of\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003eChina\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026nbsp;(No.\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e508-2043140\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e).\u003c/span\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cspan data-contrast=\"none\"\u003eAvailability of data and materials\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cspan data-contrast=\"none\"\u003eThe datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.\u003c/span\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cspan data-contrast=\"none\"\u003eEthics approval and consent to participate\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cspan data-contrast=\"none\"\u003eEthics approval was obtained from the Medical Ethics Committee of Sichuan Provincial\u0026nbsp;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003eWomen\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026rsquo;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003es and Children\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u0026rsquo;\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003es\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003eHospital.\u003c/span\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cspan data-contrast=\"none\"\u003eConsent for publication\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cspan data-contrast=\"none\"\u003eNot applicable.\u003c/span\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cspan data-contrast=\"none\"\u003eCompeting interests\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cspan data-contrast=\"none\"\u003eAll authors have declared that they have no competing interests.\u003c/span\u003e\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003e\u003cspan lang=\"EN-US\" xml:lang=\"EN-US\" data-contrast=\"none\"\u003eGDF9\u003c/span\u003e\u003cspan lang=\"EN-US\" xml:lang=\"EN-US\" data-contrast=\"none\"\u003e: Growth differentiation factor 9;\u0026nbsp;\u003c/span\u003e\u003cspan lang=\"EN-US\" xml:lang=\"EN-US\" data-contrast=\"none\"\u003eBMP15\u003c/span\u003e\u003cspan lang=\"EN-US\" xml:lang=\"EN-US\" data-contrast=\"none\"\u003e:\u0026nbsp;\u003c/span\u003e\u003cspan lang=\"EN-US\" xml:lang=\"EN-US\" data-contrast=\"none\"\u003eB\u003c/span\u003e\u003cspan lang=\"EN-US\" xml:lang=\"EN-US\" data-contrast=\"none\"\u003eone morphogenetic protein 15; POR:\u0026nbsp;\u003c/span\u003e\u003cspan lang=\"EN-US\" xml:lang=\"EN-US\" data-contrast=\"none\"\u003eP\u003c/span\u003e\u003cspan lang=\"EN-US\" xml:lang=\"EN-US\" data-contrast=\"none\"\u003eoor ovarian response; FF:\u0026nbsp;\u003c/span\u003e\u003cspan lang=\"EN-US\" xml:lang=\"EN-US\" data-contrast=\"none\"\u003eF\u003c/span\u003e\u003cspan lang=\"EN-US\" xml:lang=\"EN-US\" data-contrast=\"none\"\u003eollicle fluid; GCs:\u0026nbsp;\u003c/span\u003e\u003cspan lang=\"EN-US\" xml:lang=\"EN-US\" data-contrast=\"none\"\u003eG\u003c/span\u003e\u003cspan lang=\"EN-US\" xml:lang=\"EN-US\" data-contrast=\"none\"\u003eranulosa cells; IVF-ET:\u003c/span\u003e\u003cspan lang=\"EN-US\" xml:lang=\"EN-US\" data-contrast=\"none\"\u003eI\u003c/span\u003e\u003cspan lang=\"EN-US\" xml:lang=\"EN-US\" data-contrast=\"none\"\u003en vitro\u003c/span\u003e\u003cspan lang=\"EN-US\" xml:lang=\"EN-US\" data-contrast=\"none\"\u003e\u0026nbsp;fertilization and embryo transfer; ELISA:\u0026nbsp;\u003c/span\u003e\u003cspan lang=\"EN-US\" xml:lang=\"EN-US\" data-contrast=\"none\"\u003eE\u003c/span\u003e\u003cspan lang=\"EN-US\" xml:lang=\"EN-US\" data-contrast=\"none\"\u003enzyme\u003c/span\u003e\u003cspan lang=\"EN-US\" xml:lang=\"EN-US\" data-contrast=\"none\"\u003e-\u003c/span\u003e\u003cspan lang=\"EN-US\" xml:lang=\"EN-US\" data-contrast=\"none\"\u003elinked immunosorbent assay;\u0026nbsp;\u003c/span\u003e\u003cspan lang=\"EN-US\" xml:lang=\"EN-US\" data-contrast=\"none\"\u003eqRT\u003c/span\u003e\u003cspan lang=\"EN-US\" xml:lang=\"EN-US\" data-contrast=\"none\"\u003e-PCR:\u0026nbsp;\u003c/span\u003e\u003cspan lang=\"EN-US\" xml:lang=\"EN-US\" data-contrast=\"none\"\u003eQ\u003c/span\u003e\u003cspan lang=\"EN-US\" xml:lang=\"EN-US\" data-contrast=\"none\"\u003euantitative real-time polymerase chain reaction; AFC:\u0026nbsp;\u003c/span\u003e\u003cspan lang=\"EN-US\" xml:lang=\"EN-US\" data-contrast=\"none\"\u003eA\u003c/span\u003e\u003cspan lang=\"EN-US\" xml:lang=\"EN-US\" data-contrast=\"none\"\u003entral follicle count; CLBR:\u0026nbsp;\u003c/span\u003e\u003cspan lang=\"EN-US\" xml:lang=\"EN-US\" data-contrast=\"none\"\u003eC\u003c/span\u003e\u003cspan lang=\"EN-US\" xml:lang=\"EN-US\" data-contrast=\"none\"\u003eumulative live birth rate; DOR:\u0026nbsp;\u003c/span\u003e\u003cspan lang=\"EN-US\" xml:lang=\"EN-US\" data-contrast=\"none\"\u003eD\u003c/span\u003e\u003cspan lang=\"EN-US\" xml:lang=\"EN-US\" data-contrast=\"none\"\u003eecreased ovarian reserve; OSFs: Oocyte-secreted factors; TGF\u0026beta;:\u0026nbsp;\u003c/span\u003e\u003cspan lang=\"EN-US\" xml:lang=\"EN-US\" data-contrast=\"none\"\u003eT\u003c/span\u003e\u003cspan lang=\"EN-US\" xml:lang=\"EN-US\" data-contrast=\"none\"\u003eransforming growth factor \u0026beta;; POI:\u0026nbsp;\u003c/span\u003e\u003cspan lang=\"EN-US\" xml:lang=\"EN-US\" data-contrast=\"none\"\u003eP\u003c/span\u003e\u003cspan lang=\"EN-US\" xml:lang=\"EN-US\" data-contrast=\"none\"\u003eremature ovarian insufficiency;\u0026nbsp;\u003c/span\u003e\u003cspan lang=\"EN-GB\" xml:lang=\"EN-GB\" data-contrast=\"none\"\u003eCOS:\u003c/span\u003e\u003cspan lang=\"EN-US\" xml:lang=\"EN-US\" data-contrast=\"none\"\u003e\u0026nbsp;C\u003c/span\u003e\u003cspan lang=\"EN-GB\" xml:lang=\"EN-GB\" data-contrast=\"none\"\u003eontrolled\u003c/span\u003e\u003cspan lang=\"EN-GB\" xml:lang=\"EN-GB\" data-contrast=\"none\"\u003e\u0026nbsp;ovarian stimulation; ICSI:\u0026nbsp;\u003c/span\u003e\u003cspan lang=\"EN-US\" xml:lang=\"EN-US\" data-contrast=\"none\"\u003eI\u003c/span\u003e\u003cspan lang=\"EN-GB\" xml:lang=\"EN-GB\" data-contrast=\"none\"\u003entracytoplasmic\u003c/span\u003e\u003cspan lang=\"EN-GB\" xml:lang=\"EN-GB\" data-contrast=\"none\"\u003e\u0026nbsp;sperm injection;\u0026nbsp;\u003c/span\u003e\u003cspan lang=\"EN-US\" xml:lang=\"EN-US\" data-contrast=\"none\"\u003eFSH:\u0026nbsp;\u003c/span\u003e\u003cspan lang=\"EN-US\" xml:lang=\"EN-US\" data-contrast=\"none\"\u003eF\u003c/span\u003e\u003cspan lang=\"EN-US\" xml:lang=\"EN-US\" data-contrast=\"none\"\u003eollicle stimulating hormone;\u0026nbsp;\u003c/span\u003e\u003cspan lang=\"EN-GB\" xml:lang=\"EN-GB\" data-contrast=\"none\"\u003ehMG\u003c/span\u003e\u003cspan lang=\"EN-GB\" xml:lang=\"EN-GB\" data-contrast=\"none\"\u003e: human menopausal\u0026nbsp;\u003c/span\u003e\u003cspan lang=\"EN-GB\" xml:lang=\"EN-GB\" data-contrast=\"none\"\u003egonadotrop\u003c/span\u003e\u003cspan lang=\"EN-US\" xml:lang=\"EN-US\" data-contrast=\"none\"\u003eh\u003c/span\u003e\u003cspan lang=\"EN-GB\" xml:lang=\"EN-GB\" data-contrast=\"none\"\u003ein;\u0026nbsp;\u003c/span\u003e\u003cspan lang=\"EN-US\" xml:lang=\"EN-US\" data-contrast=\"none\"\u003eu\u003c/span\u003e\u003cspan lang=\"EN-US\" xml:lang=\"EN-US\" data-contrast=\"none\"\u003eh\u003c/span\u003e\u003cspan lang=\"EN-US\" xml:lang=\"EN-US\" data-contrast=\"none\"\u003eCG\u003c/span\u003e\u003cspan lang=\"EN-US\" xml:lang=\"EN-US\" data-contrast=\"none\"\u003e: urinary human chorionic gonadotrophin; 2PN:\u0026nbsp;\u003c/span\u003e\u003cspan lang=\"EN-US\" xml:lang=\"EN-US\" data-contrast=\"none\"\u003eT\u003c/span\u003e\u003cspan lang=\"EN-US\" xml:lang=\"EN-US\" data-contrast=\"none\"\u003ewo pronuclei;\u0026nbsp;\u003c/span\u003e\u003cspan lang=\"EN-GB\" xml:lang=\"EN-GB\" data-contrast=\"none\"\u003eE\u003c/span\u003e\u003cspan lang=\"EN-GB\" xml:lang=\"EN-GB\" data-contrast=\"none\"\u003e\u003cspan data-fontsize=\"10.5\"\u003e2\u003c/span\u003e\u003c/span\u003e\u003cspan lang=\"EN-GB\" xml:lang=\"EN-GB\" data-contrast=\"none\"\u003e:\u0026nbsp;\u003c/span\u003e\u003cspan lang=\"EN-US\" xml:lang=\"EN-US\" data-contrast=\"none\"\u003eE\u003c/span\u003e\u003cspan lang=\"EN-GB\" xml:lang=\"EN-GB\" data-contrast=\"none\"\u003estradiol\u003c/span\u003e\u003cspan lang=\"EN-GB\" xml:lang=\"EN-GB\" data-contrast=\"none\"\u003e; P:\u0026nbsp;\u003c/span\u003e\u003cspan lang=\"EN-US\" xml:lang=\"EN-US\" data-contrast=\"none\"\u003eP\u003c/span\u003e\u003cspan lang=\"EN-GB\" xml:lang=\"EN-GB\" data-contrast=\"none\"\u003erogesterone\u003c/span\u003e\u003cspan lang=\"EN-GB\" xml:lang=\"EN-GB\" data-contrast=\"none\"\u003e; TT:\u0026nbsp;\u003c/span\u003e\u003cspan lang=\"EN-US\" xml:lang=\"EN-US\" data-contrast=\"none\"\u003eT\u003c/span\u003e\u003cspan lang=\"EN-GB\" xml:lang=\"EN-GB\" data-contrast=\"none\"\u003eotal\u003c/span\u003e\u003cspan lang=\"EN-GB\" xml:lang=\"EN-GB\" data-contrast=\"none\"\u003e\u0026nbsp;testosterone; PRL\u003c/span\u003e\u003cspan lang=\"EN-US\" xml:lang=\"EN-US\" data-contrast=\"none\"\u003e: P\u003c/span\u003e\u003cspan lang=\"EN-GB\" xml:lang=\"EN-GB\" data-contrast=\"none\"\u003erolactin\u003c/span\u003e\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\" data-ccp-parastyle-defn=\"{\u0026quot;ObjectId\u0026quot;:\u0026quot;e2fc9132-75ef-4f35-97fe-3b39dcb066d4|17\u0026quot;,\u0026quot;Properties\u0026quot;:[67122396,\u0026quot;\u0026quot;,134233614,\u0026quot;true\u0026quot;,201340122,\u0026quot;2\u0026quot;,201342447,\u0026quot;5\u0026quot;,268442635,\u0026quot;20\u0026quot;,335551550,\u0026quot;6\u0026quot;,335551620,\u0026quot;6\u0026quot;,469769226,\u0026quot;等线\u0026quot;,469775450,\u0026quot;EndNote Bibliography\u0026quot;,469777841,\u0026quot;等线\u0026quot;,469777842,\u0026quot;\u0026quot;,469777843,\u0026quot;等线\u0026quot;,469777844,\u0026quot;等线\u0026quot;,469777929,\u0026quot;EndNote Bibliography 字符\u0026quot;,469778129,\u0026quot;EndNoteBibliography\u0026quot;,469778324,\u0026quot;Normal\u0026quot;],\u0026quot;ClassId\u0026quot;:1179649}\" data-ccp-parastyle-linked-defn=\"{\u0026quot;ObjectId\u0026quot;:\u0026quot;e2fc9132-75ef-4f35-97fe-3b39dcb066d4|18\u0026quot;,\u0026quot;Properties\u0026quot;:[67122396,\u0026quot;\u0026quot;,134231262,\u0026quot;true\u0026quot;,134233614,\u0026quot;true\u0026quot;,201340122,\u0026quot;1\u0026quot;,201342447,\u0026quot;5\u0026quot;,469769226,\u0026quot;等线\u0026quot;,469775450,\u0026quot;EndNote Bibliography 字符\u0026quot;,469777841,\u0026quot;等线\u0026quot;,469777842,\u0026quot;\u0026quot;,469777843,\u0026quot;等线\u0026quot;,469777844,\u0026quot;等线\u0026quot;,469777929,\u0026quot;EndNote Bibliography\u0026quot;,469778129,\u0026quot;EndNoteBibliography0\u0026quot;,469778324,\u0026quot;Default Paragraph Font\u0026quot;],\u0026quot;ClassId\u0026quot;:1179649}\"\u003e Ferraretti AP, La Marca A, Fauser BC, Tarlatzis B, Nargund G, Gianaroli L,\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003eet al.\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003eESHRE consensus on the definition of 'poor response' to ovarian stimulation for in vitro fertilization: the Bologna criteria. Hum Reprod\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003e.\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003e\u0026nbsp;2011\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003e;\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003e\u0026nbsp;26:1616-24.\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n\u003cli\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003e Kolibianakis EM, Venetis CA, Diedrich K, Tarlatzis BC, Griesinger G\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003e.\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003eAddition of growth hormone to gonadotrophins in ovarian stimulation of poor responders treated by in-vitro fertilization: a systematic review and meta-analysis. Hum Reprod Update\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003e.\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003e2009\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003e;\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003e\u0026nbsp;15:613-22.\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n\u003cli\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003e Zhen XM, Qiao J, Li R, Wang LN, Liu P\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003e.\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003eThe clinical analysis of poor ovarian response in in-vitro-fertilization embryo-transfer among Chinese couples. J Assist Reprod Genet\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003e.\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003e2008\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003e;\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003e\u0026nbsp;25:17-22.\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n\u003cli\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003e Jeve YB, Bhandari HM\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003e.\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003eEffective treatment protocol for poor ovarian response: A systematic review and meta-analysis. J Hum Reprod Sci\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003e.\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003e2016\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003e;\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003e\u0026nbsp;9:70-81.\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n\u003cli\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003e Papathanasiou A\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003e.\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003eImplementing the ESHRE 'poor responder' criteria in research studies: methodological implications. Hum Reprod\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003e.\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003e2014\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003e;\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003e\u0026nbsp;29:1835-8.\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n\u003cli\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003e Poseidon G, Alviggi C, Andersen CY, Buehler K, Conforti A, De Placido G, et al. A new more detailed stratification of low responders to ovarian stimulation: from a poor ovarian response to a low prognosis concept. Fertil Steril\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003e.\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003e2016\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003e;\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003e105:1452-3.\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n\u003cli\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003e Oudendijk JF, Yarde F, Eijkemans MJ, Broekmans FJ, Broer SL\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003e.\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003eThe poor responder in IVF: is the prognosis always poor?: a systematic review. Hum Reprod Update\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003e.\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003e2012\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003e;\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003e\u0026nbsp;18:1-11.\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n\u003cli\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003e Gonzalez-Foruria I, Penarrubia J, Borras A, Manau D, Casals G, Peralta S, et al. Age, independent from ovarian reserve status, is the main prognostic factor in natural cycle in vitro fertilization. Fertil Steril\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003e.\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003e2016\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003e;\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003e106:342-7.\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n\u003cli\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003e Younis JS, Ben-Ami M, Ben-Shlomo I\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003e.\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003eThe Bologna criteria for poor ovarian response: a contemporary critical appraisal. J Ovarian Res\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003e.\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003e2015\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003e;\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003e\u0026nbsp;8:76.\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n\u003cli\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003e Yang Y, Sun X, Cui L, Sheng Y, Tang R, Wei D,\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003eet al.\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003eYounger poor ovarian response women achieved better pregnancy results in the first three IVF cycles. Reprod Biomed Online\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003e.\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003e\u0026nbsp;2016\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003e;\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003e\u0026nbsp;32:532-7.\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n\u003cli\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003e Yakin K, Oktem O, Balaban B, Urman B\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003e.\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003eBologna criteria are predictive for ovarian response and live birth in subsequent ovarian stimulation cycles. Arch Gynecol Obstet\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003e.\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003e2019\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003e;\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003e\u0026nbsp;299:571-7.\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n\u003cli\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003e Xu B, Chen Y, Geerts D, Yue J, Li Z, Zhu G,\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003eet al.\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003eCumulative live birth rates in more than 3,000 patients with poor ovarian response: a 15-year survey of final in vitro fertilization outcome. Fertil Steril\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003e.\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003e\u0026nbsp;2018\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003e;\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003e\u0026nbsp;109:1051-9.\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n\u003cli\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003e Kyrou D, Kolibianakis EM, Venetis CA, Papanikolaou EG, Bontis J, Tarlatzis BC\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003e.\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003eHow to improve the probability of pregnancy in poor responders undergoing in vitro fertilization: a systematic review and meta-analysis. Fertil Steril\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003e.\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003e2009\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003e;\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003e\u0026nbsp;91:749-66.\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n\u003cli\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003e Laissue P, Christin-Maitre S, Touraine P, Kuttenn F, Ritvos O, Aittomaki K, et al. Mutations and sequence variants in GDF9 and BMP15 in patients with premature ovarian failure. Eur J Endocrinol\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003e.\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003e2006\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003e;\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003e154:739-44.\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n\u003cli\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003e Otsuka F, McTavish KJ, Shimasaki S\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003e.\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003eIntegral role of GDF-9 and BMP-15 in ovarian function. Mol Reprod Dev\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003e.\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003e2011\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003e;\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003e\u0026nbsp;78:9-21.\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n\u003cli\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003e Wang TT, Ke ZH, Song Y, Chen LT, Chen XJ, Feng C, et al. Identification of a mutation in GDF9 as a novel cause of diminished ovarian reserve in young women. Hum Reprod\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003e.\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003e2013, 28:2473-81.\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n\u003cli\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003e Chang HM, Qiao J, Leung PC\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003e.\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003eOocyte-somatic cell interactions in the human ovary-novel role of bone morphogenetic proteins and growth differentiation factors. Hum Reprod Update\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003e.\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003e2016\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003e;\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003e\u0026nbsp;23:1-18.\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n\u003cli\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003e Juengel JL, McNatty KP\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003e.\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003eThe role of proteins of the transforming growth factor-beta superfamily in the intraovarian regulation of follicular development. Hum Reprod Update\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003e.\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003e2005\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003e;\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003e\u0026nbsp;11:143-60.\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n\u003cli\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003e Russell DL, Robker RL\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003e.\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003eMolecular mechanisms of ovulation: co-ordination through the cumulus complex. Hum Reprod Update\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003e.\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003e2007\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003e;\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003e\u0026nbsp;13:289-312.\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n\u003cli\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003e Fang L, Cheng JC, Chang HM, Sun YP, Leung PC\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003e.\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003eEGF-like growth factors induce COX-2-derived PGE2 production through ERK1/2 in human granulosa cells. J Clin Endocrinol Metab\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003e.\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003e2013\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003e;\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003e\u0026nbsp;98:4932-41.\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n\u003cli\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003e Wu YT, Wang TT, Chen XJ, Zhu XM, Dong MY, Sheng JZ,\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003eet al.\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003eBone morphogenetic protein-15 in follicle fluid combined with age may differentiate between successful and unsuccessful poor ovarian responders. Reprod Biol Endocrinol\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003e.\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003e\u0026nbsp;2012\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003e;\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003e\u0026nbsp;10:116.\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n\u003cli\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003e Li Y, Li RQ, Ou SB, Zhang NF, Ren L, Wei LN,\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003eet al.\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003eIncreased GDF9 and BMP15 mRNA levels in cumulus granulosa cells correlate with oocyte maturation, fertilization, and embryo quality in humans. Reprod Biol Endocrinol\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003e.\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003e\u0026nbsp;2014\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003e;\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003e\u0026nbsp;12:81.\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n\u003cli\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003e Wu YT, Tang L, Cai J, Lu XE, Xu J, Zhu XM,\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003eet al.\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003eHigh bone morphogenetic protein-15 level in follicular fluid is associated with high quality oocyte and subsequent embryonic development. Hum Reprod\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003e.\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003e\u0026nbsp;2007\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003e;\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003e\u0026nbsp;22:1526-31.\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n\u003cli\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003e Alpha Scientists in Reproductive M, Embryology ESIGo\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003e.\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003eThe Istanbul consensus workshop on embryo assessment: proceedings of an expert meeting. Hum Reprod\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003e.\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003e2011\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003e;\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003e\u0026nbsp;26:1270-83.\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n\u003cli\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003e Haadsma ML, Groen H, Mooij TM, Burger CW, Broekmans FJ, Lambalk CB,\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003eet al.\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003eMiscarriage risk for IVF pregnancies in poor responders to ovarian hyperstimulation. Reprod Biomed Online\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003e.\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003e\u0026nbsp;2010\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003e;\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003e\u0026nbsp;20:191-200.\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n\u003cli\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003e Eichenlaub-Ritter U\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003e.\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003eOocyte ageing and its cellular basis. Int J Dev Biol\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003e.\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003e2012\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003e;\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003e\u0026nbsp;56:841-52.\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n\u003cli\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003e Shahine LK, Marshall L, Lamb JD, Hickok LR\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003e.\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003eHigher rates of aneuploidy in blastocysts and higher risk of no embryo transfer in recurrent pregnancy loss patients with diminished ovarian reserve undergoing in vitro fertilization. Fertil Steril\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003e.\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003e2016, 106:1124-8.\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n\u003cli\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003e Franasiak JM, Forman EJ, Hong KH, Werner MD, Upham KM, Treff NR,\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003eet al.\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003eThe nature of aneuploidy with increasing age of the female partner: a review of 15,169 consecutive trophectoderm biopsies evaluated with comprehensive chromosomal screening. Fertil Steril\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003e.\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003e\u0026nbsp;2014, 101:656-63.\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n\u003cli\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003e Hussein TS, Froiland DA, Amato F, Thompson JG, Gilchrist RB\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003e.\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003eOocytes prevent cumulus cell apoptosis by maintaining a morphogenic paracrine gradient of bone morphogenetic proteins. J Cell Sci\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003e.\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003e2005\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003e;\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003e\u0026nbsp;118:5257-68.\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n\u003cli\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003e Hobeika E, Armouti M, Kala H, Fierro MA, Winston NJ, Scoccia B,\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003eet al.\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003eOocyte-\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003es\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003eecreted\u0026nbsp;\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003ef\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003eactors\u0026nbsp;\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003es\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003eynergize\u0026nbsp;\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003ew\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003eith FSH to\u0026nbsp;\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003ep\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003eromote\u0026nbsp;\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003ea\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003eromatase\u0026nbsp;\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003ee\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003expression in\u0026nbsp;\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003ep\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003erimary\u0026nbsp;\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003eh\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003euman\u0026nbsp;\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003ec\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003eumulus\u0026nbsp;\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003ec\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003eells. J Clin Endocrinol Metab\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003e.\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003e\u0026nbsp;2019\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003e;\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003e\u0026nbsp;104:1667-76.\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n\u003cli\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003e Sugiura K, Su YQ, Diaz FJ, Pangas SA, Sharma S, Wigglesworth K,\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003eet al.\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003eOocyte-derived BMP15 and FGFs cooperate to promote glycolysis in cumulus cells. Development\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003e.\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003e\u0026nbsp;2007\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003e;\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003e\u0026nbsp;134:2593-603.\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n\u003cli\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003e Chang HM, Cheng JC, Klausen C, Leung PC\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003e.\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003eBMP15 suppresses progesterone production by down-regulating StAR via ALK3 in human granulosa cells. Mol Endocrinol\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003e.\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003e2013\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003e;\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003e\u0026nbsp;27:2093-104.\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n\u003cli\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003e Su YQ, Wu X, O'Brien MJ, Pendola FL, Denegre JN, Matzuk MM,\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003eet al.\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003eSynergistic roles of BMP15 and GDF9 in the development and function of the oocyte-cumulus cell complex in mice: genetic evidence for an oocyte-granulosa cell regulatory loop. Dev Biol\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003e.\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003e\u0026nbsp;2004\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003e;\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003e\u0026nbsp;276:64-73.\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n\u003cli\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003e Li R, Albertini DF\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003e.\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003eThe road to maturation: somatic cell interaction and self-organization of the mammalian oocyte. Nat Rev Mol Cell Biol\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003e.\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003e2013\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003e;\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003e\u0026nbsp;14:141-52.\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n\u003cli\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003e Zhang HH, Xu PY, Wu J, Zou WW, Xu XM, Cao XY,\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003eet al.\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003eDehydroepiandrosterone improves follicular fluid bone morphogenetic protein-15 and accumulated embryo score of infertility patients with diminished ovarian reserve undergoing in vitro fertilization: a randomized controlled trial. J Ovarian Res\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003e.\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003e\u0026nbsp;2014\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003e;\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003e\u0026nbsp;7:93.\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n\u003cli\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003e Barroso G, Barrionuevo M, Rao P, Graham L, Danforth D, Huey S,\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003eet al.\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003eVascular endothelial growth factor, nitric oxide, and leptin follicular fluid levels correlate negatively with embryo quality in IVF patients. Fertil Steril\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003e.\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003e\u0026nbsp;1999\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003e;\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003e\u0026nbsp;72:1024-6.\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n\u003cli\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003e Younis JS, Laufer N\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003e.\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003eRecombinant luteinizing hormone supplementation to recombinant follicle stimulating hormone\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003etherapy in gonadotropin releasing hormone analogue cycles: what is the evidence? Curr Med Res Opin\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003e.\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003e\u0026nbsp;2018\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003e;\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003e\u0026nbsp;34:881-6.\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n\u003cli\u003e\u003cspan data-contrast=\"none\"\u003e\u003cspan data-ccp-para=\"\"\u003e Crawford JL, McNatty KP\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u003cspan data-ccp-para=\"\"\u003e.\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u003cspan data-ccp-para=\"\"\u003eThe ratio of growth differentiation factor 9: bone morphogenetic protein 15 mRNA expression is tightly co-regulated and differs between species over a wide range of ovulation rates. Mol Cell Endocrinol\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u003cspan data-ccp-para=\"\"\u003e.\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u003cspan data-ccp-para=\"\"\u003e2012\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u003cspan data-ccp-para=\"\"\u003e;\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u003cspan data-ccp-para=\"\"\u003e\u0026nbsp;348:339-43.\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n\u003cli\u003e\u003cspan data-contrast=\"none\"\u003e\u003cspan data-ccp-para=\"\"\u003e Christoforou ER, Pitman JL\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u003cspan data-ccp-para=\"\"\u003e.\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u003cspan data-ccp-para=\"\"\u003eIntrafollicular growth differentiation factor 9: bone morphogenetic 15 ratio determines litter size in mammalsdagger. Biol Reprod\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u003cspan data-ccp-para=\"\"\u003e.\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u003cspan data-ccp-para=\"\"\u003e2019\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u003cspan data-ccp-para=\"\"\u003e;\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"none\"\u003e\u003cspan data-ccp-para=\"\"\u003e\u0026nbsp;100:1333-43.\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n\u003cli\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003e Sudiman J, Ritter LJ, Feil DK, Wang X, Chan K, Mottershead DG,\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003eet al.\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003eEffects of differing oocyte-secreted factors during mouse in vitro maturation on subsequent embryo and fetal development. J Assist Reprod Genet\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003e.\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003e\u0026nbsp;2014\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003e;\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003e\u0026nbsp;31:295-306.\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n\u003cli\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003e Hussein TS, Thompson JG, Gilchrist RB\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003e.\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003eOocyte-secreted factors enhance oocyte developmental competence. Dev Biol\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003e.\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003e2006\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003e;\u003c/span\u003e\u003c/span\u003e\u003cspan data-contrast=\"auto\"\u003e\u003cspan data-ccp-para=\"\"\u003e\u0026nbsp;296:514-21.\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"journal-of-ovarian-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jovr","sideBox":"Learn more about [Journal of Ovarian Research](http://ovarianresearch.biomedcentral.com)","snPcode":"13048","submissionUrl":"https://submission.nature.com/new-submission/13048/3","title":"Journal of Ovarian Research","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"GDF9 gene, BMP15 gene, Poor ovarian response, in vitro fertilization, Age","lastPublishedDoi":"10.21203/rs.3.rs-29352/v3","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-29352/v3","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground:\u003c/strong\u003e\u0026nbsp;Growth differentiation factor 9 (\u003cem\u003eGDF9\u003c/em\u003e)\u0026nbsp;and bone morphogenetic protein 15 (\u003cem\u003eBMP15\u003c/em\u003e)\u0026nbsp;genes\u0026nbsp;play important roles in\u0026nbsp;folliculogenesis.\u0026nbsp;Altered expression of\u0026nbsp;the two\u0026nbsp;have been found\u0026nbsp;among\u0026nbsp;patients with\u0026nbsp;poor ovarian response (POR). In this\u0026nbsp;prospective cohort study, we\u0026nbsp;have\u0026nbsp;determined the expression of\u0026nbsp;the\u0026nbsp;\u003cem\u003eGDF9\u003c/em\u003e\u0026nbsp;and\u0026nbsp;\u003cem\u003eBMP15\u003c/em\u003e\u0026nbsp;genes in follicle fluid (FF) and granulosa cells (GCs)\u0026nbsp;derived\u0026nbsp;from poor ovarian responders\u0026nbsp;grouped by\u0026nbsp;age, and explored its correlation with the outcome of\u0026nbsp;\u003cem\u003ein vitro\u003c/em\u003e\u0026nbsp;fertilization and embryo transfer (IVF-ET) treatment.\u0026nbsp;\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eMethods:\u0026nbsp;\u003c/strong\u003eA total of 196 patients with POR were enrolled from a tertiary teaching hospital.\u0026nbsp;The patients were diagnosed\u0026nbsp;by\u0026nbsp;the Bologna criteria and\u0026nbsp;sub-divided into group A (\u0026lt; 35 year old), group B (35-40 year old),\u0026nbsp;and group C (\u0026gt; 40 year old). A GnRH antagonist protocol was conducted for all patients, and FF and GCs were collected after oocyte retrieval. Expression of\u0026nbsp;the\u0026nbsp;\u003cem\u003eGDF9\u003c/em\u003e\u0026nbsp;and\u0026nbsp;\u003cem\u003eBMP15\u003c/em\u003e\u0026nbsp;genes\u0026nbsp;in the FF and GCs was determined with\u0026nbsp;enzyme-linked immunosorbent assay (ELISA), quantitative real-time polymerase chain reaction (qRT-PCR)\u0026nbsp;and Western blotting.\u0026nbsp;\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eResults:\u0026nbsp;\u003c/strong\u003eCompared with group C, groups A and B had significantly more\u0026nbsp;two pronuclei (2PN) oocytes\u0026nbsp;and\u0026nbsp;transplantable embryos,\u0026nbsp;in addition with\u0026nbsp;higher rates of implantation and clinical pregnancy\u0026nbsp;(\u003cem\u003eP\u003c/em\u003e\u0026nbsp;\u0026lt; 0.05).\u0026nbsp;The expression level of\u0026nbsp;\u003cem\u003eGDF9\u003c/em\u003e\u0026nbsp;and\u0026nbsp;\u003cem\u003eBMP15\u003c/em\u003e\u0026nbsp;genes\u0026nbsp;in the FF and GCs\u0026nbsp;differed significantly among the three groups (\u003cem\u003eP\u003c/em\u003e\u0026nbsp;\u0026lt; 0.05), showing a trend of decline\u0026nbsp;along\u0026nbsp;with age.\u0026nbsp;The ratio of GDF9/BMP15 mRNA levels were\u0026nbsp;similar\u0026nbsp;among\u0026nbsp;the three groups\u0026nbsp;(\u003cem\u003eP\u003c/em\u003e\u0026nbsp;\u0026gt; 0.05). The relative levels\u0026nbsp;of\u0026nbsp;GDF9 and BMP15\u0026nbsp;proteins\u0026nbsp;in GCs\u0026nbsp;have\u0026nbsp;correlated with the relative mRNA levels in GCs and protein concentrations\u0026nbsp;in FF (\u003cem\u003eP\u003c/em\u003e\u0026nbsp;\u0026lt; 0.05).\u0026nbsp;\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eConclusions:\u003c/strong\u003e\u0026nbsp;For poor ovarian responders, in particular those over 40, the expression of\u0026nbsp;\u003cem\u003eGDF9\u003c/em\u003e\u0026nbsp;and\u0026nbsp;\u003cem\u003eBMP15\u003c/em\u003e\u0026nbsp;is declined\u0026nbsp;along\u0026nbsp;with increased age and in accompany with poorer oocyte quality and IVF outcome, whilst\u0026nbsp;the ratio of GDF9/BMP15 mRNA levels\u0026nbsp;remained relatively\u0026nbsp;constant.\u0026nbsp;\u003c/p\u003e","manuscriptTitle":"Age-related decline in\u0026nbsp;the\u0026nbsp;expression of\u0026nbsp;GDF9\u0026nbsp;and\u0026nbsp;BMP15\u0026nbsp;genes in\u0026nbsp;follicle fluid and granulosa cells\u0026nbsp;derived\u0026nbsp;from poor ovarian responders\u0026nbsp;","msid":"","msnumber":"","nonDraftVersions":[{"code":3,"date":"2021-01-05 17:06:44","doi":"10.21203/rs.3.rs-29352/v3","editorialEvents":[{"type":"communityComments","content":0},{"type":"editorAssigned","content":"","date":"2020-12-17T00:00:00+00:00","index":"","fulltext":""},{"type":"decision","content":"Accept","date":"2020-12-17T00:00:00+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2020-12-16T23:00:00+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2020-12-16T23:00:00+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"journal-of-ovarian-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jovr","sideBox":"Learn more about [Journal of Ovarian Research](http://ovarianresearch.biomedcentral.com)","snPcode":"13048","submissionUrl":"https://submission.nature.com/new-submission/13048/3","title":"Journal of Ovarian Research","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}},{"code":2,"date":"2020-09-16 20:53:33","doi":"10.21203/rs.3.rs-29352/v2","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Minor revision","date":"2020-12-08T00:00:00+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2020-12-03T00:00:00+00:00","index":2,"fulltext":"Recommendation: Reviewer's comments unavailable due to the journal's policy.\n"},{"type":"reviewerAgreed","content":"","date":"2020-11-10T01:00:00+00:00","index":2,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2020-11-10T00:00:00+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"","date":"2020-11-10T00:00:00+00:00","index":1,"fulltext":""},{"type":"editorInvitedReview","content":"","date":"2020-11-10T00:00:00+00:00","index":1,"fulltext":"Recommendation: Reviewer's comments unavailable due to the journal's policy.\n"},{"type":"editorAssigned","content":"","date":"2020-09-14T12:00:00+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2020-09-13T12:00:00+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2020-09-13T12:00:00+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"journal-of-ovarian-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jovr","sideBox":"Learn more about [Journal of Ovarian Research](http://ovarianresearch.biomedcentral.com)","snPcode":"13048","submissionUrl":"https://submission.nature.com/new-submission/13048/3","title":"Journal of Ovarian Research","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}},{"code":1,"date":"2020-05-19 21:54:41","doi":"10.21203/rs.3.rs-29352/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major revision","date":"2020-08-19T12:00:00+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2020-07-17T12:00:00+00:00","index":2,"fulltext":"Recommendation: Reviewer's comments unavailable due to the journal's policy.\n"},{"type":"editorInvitedReview","content":"","date":"2020-07-05T12:00:00+00:00","index":1,"fulltext":"Recommendation: Reviewer's comments unavailable due to the journal's policy.\n"},{"type":"reviewerAgreed","content":"","date":"2020-07-03T12:00:00+00:00","index":2,"fulltext":""},{"type":"reviewerAgreed","content":"","date":"2020-06-29T12:00:00+00:00","index":1,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2020-06-25T12:00:00+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2020-05-15T12:00:00+00:00","index":"","fulltext":""},{"type":"submitted","content":"","date":"2020-05-14T12:00:00+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2020-05-14T12:00:00+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2020-05-14T12:00:00+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"journal-of-ovarian-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jovr","sideBox":"Learn more about [Journal of Ovarian Research](http://ovarianresearch.biomedcentral.com)","snPcode":"13048","submissionUrl":"https://submission.nature.com/new-submission/13048/3","title":"Journal of Ovarian Research","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"04db5047-d6c1-490e-af77-fae9843c939b","owner":[],"postedDate":"January 5th, 2021","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":1740807,"name":"Sexual \u0026 Reproductive Medicine"},{"id":1740808,"name":"Cancer Biology"}],"tags":[],"updatedAt":"2021-01-10T15:06:08+00:00","versionOfRecord":{"articleIdentity":"rs-29352","link":"https://doi.org/10.1186/s13048-020-00757-x","journal":{"identity":"journal-of-ovarian-research","isVorOnly":false,"title":"Journal of Ovarian Research"},"publishedOn":"2021-01-04 15:03:40","publishedOnDateReadable":"January 4th, 2021"},"versionCreatedAt":"2021-01-05 17:06:44","video":"","vorDoi":"10.1186/s13048-020-00757-x","vorDoiUrl":"https://doi.org/10.1186/s13048-020-00757-x","workflowStages":[]},"version":"v3","identity":"rs-29352","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-29352","identity":"rs-29352","version":["v3"]},"buildId":"oE6Zbj460LM0Up2FdVbMZ","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}