Impact of serum and follicular fluid kisspeptin and estradiol on oocyte maturity and endometrial thickness among unexplained infertile females during ICSI

Abstract

Objective: To relate serum and follicular fluid (FF) kisspeptin and estradiol levels in different stages of stimulation during Intracytoplasmic Sperm Injection (ICSI) with oocyte maturity and endometrial thickness among unexplained infertile females.

Methods: This cross-sectional study was carried out at the Australian Concept Infertility Medical Centre from March 2017 till March 2018. Fifty unexplained infertile females, booked for ICSI, were included in the study. Serum kisspeptin and estradiol were estimated by Enzyme-Linked Immunosorbent Assay in all four stages; 1: follicular stimulation, 2: ovulation induction, 3: oocyte pickup, and 4: embryo transfer. FF was aspirated during oocyte retrieval (stage 3) for the analysis of KP and estradiol. Pregnancy outcomes were categorized as non-pregnant, preclinical abortion, and clinical pregnancy.

Results: The age of the study subjects was 32.04 ± 2.29 (Mean±SD) years, with mean BMI of 28.51 ± 4.15 (Mean±SD) kg/m2. Mean serum kisspeptin and estradiol levels increased in all subjects as the stimulation proceeded stages 1-3; however, the mean dropped after retrieval of the oocytes (stage 4). Out of 27 female subjects who completed the cycle, 17 remained non-pregnant, 4 had preclinical abortion, and 6 acquired clinical pregnancy. The FF kisspeptin concentration was significantly higher than serum concentrations and positively correlated with serum and FF estradiol concentrations. FF-kisspeptin correlated with serum kisspeptin in Stage 3 (r = 0.930, p<0.001), maturity of oocyte (r = 0.511, p = 0.006) and endometrial thickness (r = 0.522, p = 0.005). Kisspeptin in stage 3 was also found to correlate with endometrial thickness (r = 0.527, p = 0.005) and with estradiol (r = 0.624, p = 0.001) independently.

Conclusion: Increase in serum and FF-kisspeptin and estradiol levels from stages 1 to 3, resulted in an optimum endometrial thickness, probability of fertilization of oocytes and chances of clinical pregnancy in Assisted Reproductive Techniques /ICSI cycles of unexplained infertile females.

Fig 1. Flow chart of the treatment protocol during the study period.

Stage 1: Follicular stimulation (FS) (n = 50). Down-regulation was followed by controlled ovarian stimulation (COS) by gonadotrophin injections (Only Inj Puregon) for fourteen days. The dose was titrated with respect to basal FSH and follicular monitoring by Transvaginal Ultrasound (TVS). Out of the 50 females, 44 females reached the ovarian induction stage (stage 2). Cycle of 4 females was cancelled due to inadequate response to stimulation, whereas two did not opt to continue the treatment. Stage 2: Ovulation induction (OI)(n = 44). On the confirmation of the maturity of at least 3 follicles (measuring18 mm in diameter) by TVS, ovulation was induced by injecting human chorionic gonadotrophin (hCG) 36 ± 1 hour before oocyte pick up (OPU). Maturity of follicles was not confirmed in six females, we could not perform OPU of these females. On the same day, endometrial thickness was measured by a Transvaginal Scan (TVS) in the midsagittal plane by two-dimensional ultrasound with a 7.5 MHz vaginal probe (Hitachi E UB 525; Hitachi, Tokyo Japan) at the thickest endometrial segment [17]. Stage 3: Oocyte Pick Up (OPU) (n = 38). Mature oocytes of females (n = 38) were retrieved 36 hours after hCG injection by vaginal ultrasound probe with 16G adapter and double-lumen oocyte aspiration needle on 14^th, 15^th, or 16^th day of stimulation. All the collected eggs were treated and then transferred to the incubator for about 1–2 hours prior to insemination by ICSI procedures. During this phase, follicular fluid (FF) was acquired from all follicles (measuring 18 mm or more on ultrasound) from each ovary of the patient [18]. Stage 4: Embryo transfer (ET) (n = 27). After confirmation of fertilization and cleavage (18 to 24 hours after egg collection), embryos were graded from 1–5 [19]. The embryo transfer of 27 females (blastocysts) was done on Day 5 using Sims-Wallace Embryo Replacement Catheter, under ultrasound guidance.

Fig 2

Comparison of estradiol (a) and kisspeptin (b) in all stages of stimulation. *Significant with stage 1 at 0.05 ** Significant with stage 1 at 0.01. Stage 1, FS: Follicular stimulation, Stage 2, OI: Ovulation Induction, Stage 3, OPU: Oocyte pickup, Stage 4, ET: Embryo transfer.

Fig 3

Comparison of estradiol (a) and kisspeptin (b) at various stages of stimulation in all three outcome groups. *Significant with stage 1 at 0.05 ** Significant with stage 1 at 0.01. FS: Follicular stimulation, OI: Ovulation Induction, OPU: Oocyte pickup, ET: Embryo transfer.

Fig 4

a) Correlation of Follicular estradiol and follicular KP b) Correlation of Follicular KP with number of mature oocytes c) Correlation of Follicular KP with Serum KP3 d) Correlation of Serum KP3 with Serum estradiol e) Correlation of Serum KP3 with endometrial thickness.