Dynamic Changes Of Inhibin B In Serum And Follicular Fluid And Its Effects On Controlled Ovarian Hyperstimulation Outcome

Objective: Inhibin B(INHB),a multifunctional cell growth factor of TGF-?,is a heterodimeric glycoprotein hormone built up from an ? and a?-subunit(B-type).In the ovary,INHB is mainly produced by the granulosa cells(GCs)of small and mid-follicles.INHB secreated by GCs is able to specifically act on pituitary gland and selectively inhibit the secretion of pituitary FSH by a negative feedback.In addition,it can improve the substrate of E2,then regu-late the secretion of E2 by autocrine/paracrine mechanisms.The classical endocrine and autocrine/paracrine mechanisms of INHB involved in follicular development,selection as well as oocytes maturation.In this study,the levels of serum INHB in different time nodes and diff-erent follicular sizes on oocytes retrieved day during different controlled ovarian hyperstimulation(COH)protocols were detected,and the correlation with INHB and COH outcome was analyzed.The aim of this study was to explore the dynamic changes of serum as well as FF INHB and its effects on COH outcomes.Methods:1 Study participants Seventy-six women underwent in vitro fertilization(IVF)or intracy-toplasmic sperm injiction(ICSI)-embryo transfer(ET)therapy for the first time due to the tubal or male factors from July 2015 to December 2015 in our reproductive center were included in this study.Ultimately,they were randomly grouped into a down-regulation protocol(DRP)or non-pituitary down-regulation protocol(NDRP).The following inclusion criteria were adopted: 1)less than 35 years;2)antral follicular count(AFC):10-20;3)Body mass index(BMI):18-25 kg/m2;4)regular menstrual cycles;5)normal basal hormone levels;6)no history of ovarian surgery;7)no other endocrine diseases and no use of hormone drugs in the past six months.Exclusive criteria:1)hyperprolactinemia;2)polycystic ovarian syndrome(PCOS);3)endome-triosis(EMT).This study has been approved by the hospital ethics committee and patients have signed the informed consent form.2 COH protocols2.1 Down-regulation protocol(DRP)The gonadotropin-releasing hormone agonist(Gn RH-a)triptorelin(0.01 mg for each,Ipsen Pharma Biotech,France)was started at 0.1mg/d in the mid-luteal phase of the preceding cycle.On the third day of menstrual cycle after pituitary down-regulation,serum estrogen(E2),progesterone(P),lutein-zing hormone(LH)and follicular stimulation hormone(FSH)were detected and ultrasonic monitoring were begining.When the levels of E2,P,LH and FSH were respectively less than 50 pg/ml,2 ng/ml,5 m IU/ml and 5m IU/ml,no follicles were great than 10 mm,endometrium was less than 5 mm and there was no functional cyst,triptorelin was reduced to 0.05 mg/d.Meanwhile,75-300IU/d of gonadotropins(Gn)was added until the ratio of follicles more than 18 mm to follicles more than 14 mm was greater than or equal to sixty percent and the averge level of serum E2 was 200~300 pg/ml in each follicle more than 14 mm.Then 250 ug of recombinant human chorionic gonadotropin(rh CG)was subcutaneously administered 35~36 h before transvaginal oocyte retrieval.Less than or equal to three good-quality embryos were transferred after 3 days of OUP,when endometrium was more than 8mm,the level of P was less than 1.5ng/ml and there was no risk of ovarian hyperstimulation syndrome(OHSS).On the day of OPU,patients were treated with luteal phase support with 60 mg of progesterone injection(20mg for each,Xian Ju Pharma-ceutical company,Shang Hai)and two tables/d of progesterone soft capsules from embryo transferred day.Pregnancy was first assessed using serum ?-h CG after 14 days of OPU and clinical pregnancy was defined by the presence of an intrauterine gestational sac after 4 weeks of OPU.2.2 Non-pituitary down-regulation protocol(NDRP)2.5mg/d of letrozole(LE,2.5mg for each,Heng Yuan Pharmaceutical company,Jiang Su)was taken from the third day of menstruation and 150-300IU/d of h MG was injected.After 5 days,stoped taking the LE and added50-100mg/d of clomiphene(CC,50 mg for each,Crprus High Pharmaceutical Factory)to trigger day.Serum E2,P,LH and FSH were detected.At the same time,folliclar diameters as well as counts were strictly monitored by ultrasound.Reaching to the standard of trigger,0.2mg/d of triptorelin was administered subcutaneously and 50mg/8h of indometacin suppo-sitories was rectally used 35~36h before transvaginal oocyte retrieval.The other processes in NDRP were similar to DRP.3 Serum and FF collection Blood samples were obtained on the third day of menstruation(d3),the third day of menstruation after pituitary down-regulation(d MPDR3),Gn starting day(d Gn0),the fifth day of Gn administration(d Gn5),the day of h CG administration(dh CG),the next day of h CG administration(dh CG1)and 2days after OPU(d OPU2).Serum was transferred into sterile tubes after centri-fuged at 2000×g for 10 min and frozen in aliquots at-80? for subsequent centralized assays.At the same time,FF was divided into three categories by follicular diameter during OPU.(Big follicle(BF): diameter?18mm;Middle follicle(MF): 14mm?diameter<18mm;Small follicle(SF): 10 mm?diameter<14mm).FF was transferred into sterile tubes after centrifuged at 3000×g for10 min and frozen in aliquots at-80? for subsequent centralized assays.FF contaminated with blood was excluded.4 Hormonal measurements in serum and FF INHB concentrations were measured from all serum samples and FF by enzyme-linked immunosorbent assay(Anshlabs,USA).Serum E2,P,LH and FSH were tested by chemiluminescence immunoassay kit(Elecsys,Roche,Germany).5 Statistical analysis Quantitative data results were presented as mean ± standard deviation(x±s),determined using statistical package for social science(SPSS)21.0.Comparisons of the two groups were performed using independent-sample t-test and paired-sample t-test.The correlation between variables was determ-ined by Pearson correlation coefficient.The statistical significance was set as P <0.05.Results:1 Comparison of patients' characteristics A total of 76 patients undergoing IVF were included in the study.There were 38 people in both DRP and NDRP.No statistical differentces were found in the two protocols about patients' age,menstrual cycle,years of infertility,basal hormone levels and AFC(P>0.05).Data of the two sets were comp-arable.2 Comparison of correlated parametes about COH The dosage of Gn in DRP was 1998.68±154.76 IU which significantly reduced when compared with 2110.20±150.00 IU in NDRP(P=0.002).Endo-metrium and E2 on h CG day in DRP were 10.83±1.51 mm and 4160.40±1073.67pg/ml which were remarkably higher than 9.12±0.98 mm and 3737.51±643.28pg/ml in NDRP(P=0.000;P=0.041).Compared with NDRP,retrieved oocytes was slightly higher in DRP,however,duration of Gn,number of M? oocytes,2PN fertilization,high-quality embryos and available embryos were a little lower in DRP.There were no statistical differences with them,(P>0.05).3 Dynamic changes of INHB in serum and FF during COH The levels of INHB on d MPDR3 and d Gn0 were 50.63±23.80pg/ml and28.99±8.83pg/ml,which were markedly declined when compared with the basal level of 89.07±26.07pg/ml(P=0.000).The levels of INHB on d Gn0 in DRP was 28.99±8.83pg/ml that was dramatically lower than 79.08±21.98pg/ml in NDRP(P=0.000).After exogenous stimulation,the INHB in the two protocols were begining to increase.INHB went to a peak on dh CG and then began to decline,especially on d OPU2(P<0.01).There were no statistical differentces about INHB in the two protocols after d Gn0 as well as the d3(P>0.05).FF INHB was positivly correlated with the folliclar sizes.There was no statistical difference about INHB level in BF(P>0.05).However,the levels of INHB in MF and SF were significantly higher in DRP(P=0.000).4 Correlations of serum and FF INHB level with COH outcomes The detection time of correlations between INHB and COH outcomes from strong to weak were d Gn5,dh CG,dh CG1,d3 and d OPU2 in the two protocols.At the same time,INHB on d MPDR3 and d Gn0 had good correlations with COH outcomes in DRP.INHB on d Gn0 was significantly negative with Gn dose(r=-0.716,P<0.01)and evidently positive with E2 on dh CG(r=0.600,P<0.01).In the two protocols,INHB in BF had the strongest correlation with COH outcomes.INHB in MF also had better relationship with COH outcomes.However,the correlation between INHB in SF and COH outcomes were ambiguous.Conclusion:1 The levels of INHB during down-regulation in the down-regulation protocol were significantly decreased,and the level after down-regulation indicates the better result of follicular synchronization with down-regulation.2 Levels of INHB in serum,big follicles as well as in middle follicles are good markers to predict ovarian response and COH outcomes,but the level of INHB in small follicles had little correlation with COH outcome.