Animals
ICR female mice, aged 3-5 weeks were used for this experiment, which were purchased from the company (Beijing Vital River Laboratory Animal Technology Co., Ltd. China). All animals were maintained under regulated 12h light on/12h light off cycles and were fed with standard mice chow and water ad libitum.
Progestin-Primed Ovarian Stimulation Protocol in mice
Mouse in diestrus of estrous cycle were chosen to induce COH. In order to simulate PPOS protocol, mice (30g ±2g) were intraperitoneally injected with 10 IU pregnant mare serum gonadotropin (PMSG) and progesterone (2mg/kg , 5mg/kg, dissolved in corn oil) at first day, while the mice of control group were only intraperitoneally injected with 10 IU PMSG and corn oil. After 24h, the progesterone oil agent and oil was injected in PPOS group and control group respectively. After 48 h, the mice were given an intraperitoneal injection with 10 IU human chorionic gonadotropin (HCG) to induce the ovulation in the PPOS and control groups.
Oocyte isolation and Detection of Ovulation
Oocytes were collected in oviducts and ovaries at 12h, 12.5h, 13h, 14h and 16h after HCG injection. Oocytes from oviducts were also collected by puncturing the ampulla of oviducts. Cumulus-cell-oocyte complexes (COCs) were isolated from large antral follicles (preovulatory follicles, containing an oocyte and the diameter of follicles >400um 23) in ovaries by needle puncture under a dissecting microscope. Removal of cumulus granulosa cells by repeated blowing and aspiration of COCs using glass pipette with adding traces of hyaluronidase enzyme into the medium. The medium used for COCs and oocytes was the modified HTF medium supplemented with 5% serum substitute supplement (FUJIFILM Irvine Scientific). Meanwhile, 5M IBMX (Sigma) was added for preventing oocytes from further mature. Oocytes isolated from ovaries may have four different developmental stages which include the germinal vesicle (GV), GV breakdown (GVBD), metaphase I (MI) and metaphase II (MII). Oocytes in different developmental stages were counted respectively, and their morphological appearance was photographed using an Olympus IX71 inverted microscope.
Hormone measurements
To determine estradiol (E2), progesterone (P) and LH levels, the whole blood of mice was collected at different hours after HCG injection. For LH testing, whole 6μL blood was immediately diluted in 114 μL of 0.1M PBS with 0.05% Tween 20, vortexed, and snap frozen on dry ice. Samples were stored at -20°C for a subsequent LH ELISA 24,25. For estradiol (E2) and progesterone (P) testing, serum was isolated from whole blood by centrifuge at 3000 rpm for 10 minutes and cryopreserved at -80°C until test. Estradiol (E2) and progesterone (P) levels were examined by the laboratory of Shanghai Ninth People's Hospital, using chemiluminescence (Abbott Biologicals B.V.).
Hematoxylin-eosin staining and Immunofluorescence
Mice were injected anesthetic solution (ketamine: xylazine) with no more than 200 µL dose. Then mice died within 5 seconds after stopping the injection and perfused with saline followed by 4% paraformaldehyde in 0.1M PBS. The ovaries were quickly removed, post-fixed in 4% paraformaldehyde overnight, and subjected to dehydration in increasing saccharin solutions (20–30%) at 4°C. The frozen ovary slices were sectioned at 10 µm on a vibratome (Leica) for hematoxylin and eosin (H&E) staining. Preovulatory follicle was defined as containing an oocyte and the diameter of follicles >400um 23, and corpus luteum were defined as cellular and/or partially eosinophilic fluid-filled spaces lined by polygonal eosinophilic cells containing lipid droplets 23,26. Images were captured on an olympus fluorescence microscope (BX53).
For immunofluorescence, the ovaries slices were washed three times with PBS, then separately incubated with the first antibody (anti-HCG receptor, PA1552, BOSTER; anti-cyclooxygenase 2, ab15191, Abcam; anti-progesterone receptor, ab16661, Abcam; anti-PPARγ, E8-sc-7273, Santa Cruz Biotechnology; anti-VEGF, C-1-sc-7269, Santa Cruz Biotechnology; anti-HIF-1α, NB100-105, Novus Biologicals; anti-ADAMTS-1, PA5-47790, Invitrogen) with suggested dilution at 37°C 2h. All primary antibodies were supplemented with 0.3% TritonX-100 and 3 % equinum serum in PBS. After washing three times with PBS, ovary slices were stained with the Alexa Fluor 488 secondary antibodies respectively at 37°C 1h. Then ovary slices were washed three times with PBS and stained with DAPI at 37°C for 10 minutes, and mounted after rinsing. Fluorescence images were captured on an Olympus FV3000 confocal microscope.
RNA Extraction and Real-Time PCR
Mouse ovaries were isolated, frozen in TRIzol Reagent (Shanghai Pufei Biotech Co., Ltd) and stored at –70 °C until extraction. Total RNA was extracted from ovaries using the TRIzol Reagent according to the manufacturer’s instructions, with the inclusion of a DNase digestion step. RNA concentrations were quantified using the Nano pro (Nano Drop Technologies, Wilmington, DE). The Superscript First-Strand Synthesis System for reverse transcription (RR037A,PrimeScript™ RT reagent Kit, TAKARA) was used with random primers. For amplification of the cDNA products, specific primers pairs were selected as indicated in Additional file 1. The target cDNA was amplified by gene-specific primers and SYBR (TB Green Premix Ex Taq, TAKARA), and carried out on an ABI-PRISM 5700 sequence detection system. The mRNA expression of target gene was normalized to the internal control ribosomal protein L-19 (L19) .
Clinical Study setting and patients
The retrospective study was conducted between January 2015 and Dec 2021 in the Department of Assisted Reproduction of the Ninth People’s Hospital affiliated with Shanghai Jiao Tong University School of Medicine. The first IVF/ICSI cycle of patients (n=11997) were included: female age less than 40 years; basal follicle stimulating hormone (FSH) level<10 mIU/mL; and antral follicle count (AFC) ≥5. The following exclusion criteria were applied: 1) patients who had endometriosis or polycystic ovarian syndrome, 2) patients who received hormone treatments within the previous three months; and 3) any contraindications to ovarian stimulation.
Clinical Ovarian Stimulation (COS) Protocols
In the PPOS protocol, hMG150–225 IU/d and medroxyprogesterone acetate (MPA, 10 mg/d, XianJu Pharmaceutical Co., Zhejiang, China) were administered from menstruation cycle days 2–4. All the doses were adjusted in line with the transvaginal ultrasound examination outcomes. Final oocyte maturation was by Triptorelin 0.1-0.2 mg (Ferring Pharmaceuticals, Germany) and/or HCG 1000-5000 IU (Lizhu Pharma ceutical Trading Co., Shanghai, China) when three dominant follicles reached at least 18 mm or one dominant follicle reached at least 20 mm in diameter.
Clinical Oocyte retrieval operation and insemination
All available follicles > 10 mm in diameter were aspirated by skilled physicians using transvaginal ultrasound–guided follicle aspiration method after trigger. The timing of oocyte aspiration was determined as the midpoint of the start time (patient transferred to the operating table) and end time (patient removed from the operating table), which were recorded in the system. The oocytes were then transmitted to the modified HTF medium (Irvine Scientific, USA) and transferred to culture medium. Fertilization was performed by IVF or ICSI, which depends on semen parameters. Oocyte maturity was determined after preincubating 2-3h and then denuding in ICSI; however, maturity was examined the day after the retrieval day in IVF. This study was exempted from Institutional Review Board (IRB) approval because of its retrospective nature and the use of anonymous database.
Statistical analysis
The statistical analyses in mice were performed in the GraphPad Prism (GraphPad Software). When comparing the concentration of serum hormones at different timepoints within two groups, data were analyzed by Mann-Whitney test. The number of oocytes, ovulated oocytes and ovulated MII were also analyzed by Mann-Whitney test. When comparing percentage of oocytes in ovary, the rates of ovulation and the ovulation rates of MII, chi-square test was used. When comparing the mRNA expression level of specific genes within two groups, Mann-Whitney test was used. Differences were considered significant when p<0.05. Values are given as mean ± SEM.
The clinical statistical analysis was conducted by R studio (version 3.6.3). The definitions used in this study are follows: oocyte retrieval rate = the number of cumulus–oocyte–complex (COC) retrieved/the number of follicles ≥10 mm present on the retrieval day; mature oocyte rate = the number of mature oocyte / the number of oocytes retrieved. Differences were considered significant when p<0.05.