A total of 80 patients who underwent IVF-ET between July 2017 and August 2018 in the Center for Reproductive Medicine, Third Affiliated Hospital of Guangzhou Medical University, were recruited for this study. The study population consisted of Forty women with EM and 40 women without EM. The presence or absence of endometriosis was conﬁrmed by laparoscopic surgery and post-operative histological examination. Women with polycystic ovary syndrome, diabetes, hypertension, dyslipidemia, HIV infection or any active infection and autoimmune diseases were excluded. Follicular fluid (FF) was extracted from both groups.
Women were monitored and managed according to the hospital’s clinical protocols. Various controlled ovarian stimulation (COS) protocols were used, with 150-450 IU/day of recombinant FSH or human menopausal gonadotropin in a gonadotropin-releasing hormone antagonist protocol, a long agonist protocol, or a short agonist protocol. The protocols were determined according to each patient's characteristics (age, body mass index (BMI), AFC and AMH). Transvaginal oocyte retrieval was scheduled 35-36 h after hCG injection ART was performed per standard operating procedure of the hospital. Fertilization was assessed by the appearance of two pronuclei. Cleavage stage embryos were graded as per the Istanbul consensus. Fresh embryo transfer (ET) was performed 2-3 or 5 days later. Embryos were vitrified frozen on day 3,5 or 6. The luteal phase was supported by vaginal administration of micronized progesterone (400 mg/d) started on the day of ovarian puncture.
Follicular fluid collection
Follicular fluid was preserved at oocyte retrieval, by collecting the liquid aspirated from the follicle into the suction tube, to avoid contamination by blood. Follicular fluid samples from each follicle were pooled for each patient for measurement of KS concentrations. Pooled follicular fluid was centrifuged at 1500g for 10 min to eliminate cells and cell debris. And the supernatants were then stored at -20°C for KS concentration measurements.
KS and hormone assays
The follicular fluid KS concentrations were collected and determined measured by enzyme-linked immunosorbent assay (human kallistatin ELISA kits, Catalog Number: DY1669; R&D Systems, USA) according to the manufacturer's protocol. The concentrations of serum oestradiol, FSH, LH, progesterone, testosterone and AMH, and follicular fluid oestradiol and progesterone, were measured by chemiluminescence (Roche, Switzerland).
The reproductive outcome of this study included implantation, clinical pregnancy, live birth rate (LBR) and spontaneous miscarriage rates. Neonatal outcomes included preterm birth, stillbirth, birth weight, low birth weight and congenital anomalies. Live birth was defined as the delivery of any viable neonate who was 28 weeks of gestation or older, and twins delivered by one mother were calculated as one live birth. Clinical pregnancy was defined as the present of gestational sac on ultrasound at 6-8 weeks of gestation; low birth weight was defined as the birth weight less than 2500 g and very low birth weight less than 1500 g.
The statistical analysis was performed using the Statistical Package for Social Science (SPSS) version 22.0. The baseline characteristic was expressed as the mean ± SD (standard deviation) and differences in variables were compared by means of Student’s t-test. Categorical variables were described as frequencies and percentages, and compared using chi-square test. A P-value of 0.05 was considered significant.