Correlation between steroid levels in follicular fluid and hormone synthesis related substances in its exosomes and embryo quality in patients with polycystic ovary syndrome

Samples were collected from September 2019 to November 2019. Patients have given their written informed consent. The study was approved by the Ethical Committee of the Zhongshan Hospital, Fudan University (Shanghai, China), and carried out in compliance with the Population and Family Planning Law of the People’s Republic of China (The ethical approval number: B2018–244).

The diagnosis of PCOS has based on the 2003 Rotterdam diagnostic criteria which require two of the following three manifestations: (1) oligo- and/or anovulation, (2) clinical and/or biochemical evidence of hyperandrogenism, and (3) polycystic ovaries on ultrasound examination (at least 10 follicles 2–9 mm in size or volume of the ovary greater than 10 mL) [3]. Ten patients were selected as the PCOS group who require the three manifestations at the same time and underwent in vitro fertilization (IVF) with intracytoplasmic injection (ICSI) at Reproductive Center, Zhongshan Hospital, Fudan University. Another ten women who underwent IVF treatment with an indication of male factor infertility during the same period were selected as the control group. All the recruited patients were under 36 years old and had a normal BMI range from 18.8 to 25 kg/m². All patients received the antagonist stimulation protocol as reference [24].

The BMI values, hormonal, biochemical and ultrasonographic parameters of the control and PCOS patients were documented. BMI (kg/m²) was calculated as the ratio of the weight (kg) to the square of the height (m²). Patients received a standard in vitro fertilization (IVF) flexible-start antagonist stimulation protocol with exogenous FSH and human menopausal gonadotropins at individualized doses and underwent transvaginal ultrasound-guided follicle aspiration 36 h after human chorionic gonadotropin (hCG) and/or a GnRH agonist.

The follicular fluid of the largest, first punctured follicle was collected during the oocyte retrieval procedure (transvaginal follicular aspiration). All the follicles we selected are surrounded by the cumulus-oocyte complexes. Each ovarian follicle was aspirated independently. The collected FF was checked for red blood cells. FF contaminated with red blood cells was excluded from the study.

All oocytes retrieved from the FF samples were cultured and fertilized in a separate culture dish for evaluating the quality and embryo development of oocytes. Normal fertilization was defined by the presence of two pronuclei (2PN). A top-quality embryo was defined as an embryo with (i) seven or more blastomeres on day 3, (ii) 20% fragmentation or less on day 3, and (iii) no multinucleated blastomeres ever as described by Van Royen et al. [25]. On the fifth day of the culture, at the blastocyst stage, three criteria were taken into consideration: the development of inner cell mass (ICM), the appearance of trophectoderm (TE) and the expansion of the blastocyst cavity. Blastocyst Day 5–6 on was considered to be a fully expanded (Grade 4) through to a hatched (Grade 5) blastocyst with a high-quality ICM (Grade 1) and TE (Grade 1) [26].

Twenty steroids were measured in 200 μl of follicular fluid samples using LC-MS/MS. Details of the LC- MS/MS methods are presented in the reference [14]. The panel included progestogens (including mineralocorticoids and glucocorticoids), androgens and estrogens biosynthesized in steroid metabolic pathways. Because of the high concentrations of 17-OH pregnenolone, progesterone, estradiol and estrone, follicular fluid samples were run a second time diluted 100-fold with double-stripped, delipidated normal human plasma (blank matrix plasma, Product code: 1800–0058, SeraCare Life Sciences, MA, USA) to quantify those three steroids.

The follicular fluid exosomes were isolated by ultracentrifugation. Follicular fluid was centrifuged at 300 g for 10 min to remove cells. The supernatant fluid was then centrifuged at 2000 g for 10 min at 4 °C to remove dead cells. The resultant supernatant fluid was transferred to an ultracentrifuge tube and centrifuged at 100000 g for 2 h. The pellet was suspended in PBS and filtered through a 0.22-μm filter, and then centrifuged at 100000 g for 2 h. The pellet was resuspended in 200 μL PBS and stored at − 80 °C.

A total of 20 μL of exosome suspension (5 μg/μL) was fixed on a continuous grid and then negatively stained with 2% uranyl acetate solution for 1 min and air-dried. The samples were observed by FEI Tecnai G2 spirit transmission electron microscope (FEITM) at an acceleration voltage of 120 kV.

Nanoparticle tracking analysis (NTA) measurements were performed using a NanoSight NS300 instrument (Malvern Panalytical) with a 488-nm laser and sCMOS camera module (Malvern Panalytical). Measurements in flow mode were performed with a flow rate of 50, these flow measurements consisted of 3 measurements of 60 s, and the captured data were analysed using NTA 3.2 software.

The concentration of proteins was quantified using the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific) following the manufacturer’s instructions. Proteins were separated by 12% SDS-PAGE gels and transferred to PVDF membranes by gel electrophoresis and electroblotting, respectively. After blocking with 5% BSA, blots were probed with primary antibodies at 4 °C overnight. Then, membranes were washed and incubated with secondary antibodies. Ultimately, proteins were visualized using the enhanced chemiluminescence reagents (Thermo Fisher Scientific). CD9 antibody: CD9 (D8O1A) Rabbit mAb (#13174, Cell signaling technology); CD63 antibody: CD63 (D4I1X) Rabbit mAb (#55051, Cell signaling technology); TSG101 (ab125011, abcam); Calnexin (#2433, Cell signaling technology).

Total RNA was extracted using TRIzol (Invitrogen), and RNA was then reverse-transcribed using SuperScript First-Strand cDNA System (Takara) according to the manufacturer’s instructions. Quantitative RT-PCR (qRT-PCR) was performed using the SYBR Green PCR master mix (Takara) and the StepOnePlus PCR system (Thermo Fisher Scientific) according to the manufacturer’s instructions. The house- keeping gene GAPDH was used as an endogenous control. The primer sequences are shown in Table S3.

This study included ten PCOS patients and ten male-factor infertility patients. The demographic features, biochemical, and hormonal data of PCOS group and normal group are showed in Table 1. No statistically significant differences were found between the groups in relation to age, BMI, basal FSH, LH, estradiol and progesterone levels. Baseline testosterone was higher in PCOS women. Furthermore, more oocytes and less MII oocytes were retrieved in the PCOS women. The rate of retrieved top-quality embryos and embryos develop to blastocyst were significantly lower in women with PCOS. These data indicate that the quality of oocyte and embryo tend to be lower in PCOS patients during assisted conception, which may be linked to metabolism-induced changes in the oocyte through the microenvironment of follicular fluid (FF).

To evaluate the possibility of using key reproductive hormones as potential indicators of oocytes development, 20 steroids were quantified and compared between PCOS patients and controls by LC-MS/MS. The data displayed in Table S1 show that the control group had lower pregnenolone levels in follicular fluid (mean, 123.91 ± 15.93 ng/mL) than the PCOS women (mean, 245.04 ± 33.40 ng/ml) and the differences were statistically significant (p = 0.006). Progesterone levels in follicular fluid were higher in normal group (mean, 12,590 ± 1393.59 ng/ml) than they were in PCOS (mean, 9153.38 ± 542.39 ng/ml) (p = 0.041). FF estriol levels were significantly increased (P = 0.003) in PCOS group (7.66 ± 0.76 ng/ml) compared to the normal group (4.60 ± 0.49 ng/ml) (Table S1, Fig. 1). Similarly, FF estradiol levels were significantly higher (P = 0.047) in PCOS group (546.32 ± 68.95 ng/ml) compared to normal group (374.38 ± 41.92 ng/ml) (Fig. 1).

We further elucidated whether the estrogen and progestogen level predicted the outcome of ovarian stimulation and embryo quality. Our results showed that, with increasing FF estriol levels, significantly less MII oocytes (r = − 0.576, p = 0.008) (Fig. 2a) were obtained in PCOS women, and tended to have a reduced chance to develop into a morphological top-quality embryo (r = − 0.545, p = 0.013) (Fig. 2b). Moreover, follicular fluid estriol levels were negatively correlated with rate of blastocysts (r = − 0.525, p = 0.017) (Fig. 2c). Increased estradiol levels in follicular fluid were associated with a lower rate of MII oocytes (r = − 0.462, p = 0.04) (Fig. 2d). Furthermore, pearson correlation analysis showed that FF levels of pregnenolone were negatively and significantly correlated with top-quality embryo (r = − 0.476, p = 0.034) (Fig. 2h) and rate of embryos develop to blastocysts (r = − 0.698, p = 0.001) (Fig. 2i). Progesterone level was not significantly related with embryo quality (Fig. 2j & k & l). This may be the evidence of the immaturity of follicles as well as negative effects of estriol and estradiol on folliculogenesis and oocyte quality in PCOS patients.

Human FF exosomes were characterized with regard to their morphology, diameter and the presence of exosome-enriched protein markers. Electron microscopic images (Fig. 3a) showed that extracted extracellular vesicles (EVs) were cup-shaped structures with a diameter of about 30–150 nm. Moreover, the presence of the exosome markers CD63, CD9 and TSG101 proteins were confirmed by western blot (Fig. 3b). The size distribution measured by NTA also showed a typical profile of exosomes (Fig. 3c). TEM, NTA and Western blot results were consistent with the characteristics of exosomes.

The mRNA expression levels of these genes from the FF exosomes were examined in our study, and the results showed significantly increased CYP11A, CYP19A and HSD17b1 mRNA expression in the PCOS group (Fig. 4), indicating that the increase in E2 was associated with increased expression of key genes involved in hormone synthesis. Whereas STAR and HSD3b2 mRNA expression were not affected (p > 0.05). CYP19A1 encodes cytochrome P450 aromatase that converts androgens to 17β estradiol [27]. HSD17B1 is expressed in the syncytiotrophoblast cells of the placenta [28] and ovarian granulosa cells [29], and both of these cell types are involved in de novo estrogen biosynthesis. CYP11A1 is the gene encoding for the key enzyme in metabolism of cholesterol to pregnenolone. The increased mRNA expression on exosome in FF is consistent to the increased pregnenolone level in FF in PCOS patients. The increases in the expression levels of HSD17B2, CYP11A and CYP19A were accompanied by the changes in hormonal levels in FF, indicating a condition shifting from the progesterogenic follicles to estrogenic follicles in PCOS patients.

We further elucidated whether CYP11A, CYP19A and HSD17b1 mRNA level predicted the hormone in FF and the outcome of ovarian stimulation and embryo quality. Pearson correlation analysis (Fig. 5) showed that CYP11A mRNA level was positively and significantly correlated with estradiol, estriol and pregnenolone levels. The elevated CYP19A mRNA level was coraleted with estradiol. There is a positive correlation between HSD17b1 mRNA in exosomes from FF and estradiol and pregnenolone was significant.

Furthermore, pearson correlation analysis showed that exosomal CYP11A mRNA level were negatively and significantly correlated with top-quality embryo (r = − 0.768, p < 0.01) (Fig. 6b) and rate of embryos develop to blastocysts (r = − 0.691, p < 0.01) (Fig. 6c). Our results also showed that, with increasing exosomal CYP19A mRNA levels, significantly less MII oocytes (r = − 0.506, p = 0.023) (Fig. 6d) were obtained in women, and tended to have a reduced chance to develop into a morphological top-quality embryo (r = − 0.819, p < 0.01) (Fig. 6e). Moreover, exosomal CYP19A mRNA levels were negatively correlated with rate of blastocysts (r = − 0.528, p = 0.017) (Fig. 6f). Increased HSD17b1 mRNA levels in follicular fluid were associated with a lower rate of top-quality embryo (r = − 0.796, p < 0.01) (Fig. 6h) and blastocysts (r = − 0.784, p < 0.01) (Fig. 6i). There is a negative correlation between CYP11A, CYP19A and HSD17b1 mRNA level in exosomes in follicular fluid and oocyte quality.