| IntroductionControlled ovarian hyperstimulation(COH)plays a key role in in vitro fertilization/intracytoplasmic sperm injection-embryo transfer(IVF/ICSI-ET), comparing with ideal retrieved oocytes,the average pregnancy rate stays low.One of the possible responsible mechanisms is during COH, substantial sex hormone were produced,resulting in hypercoagulability causing thrombosis of maternal vessels with reduced perfusion of the intervillous space and placentation failure.furthermore unsuccessful implantation or placentation. State of hypercoagulability associated with hormonal milieu resulting from the use of contraceptive pills or pregnancy has been documented. During COH supra-physiological estrogen and progesterone were produced,which changed the state of coagulation and fibrinolytic system, causing a hypercoagulable state.Present studies investigating fibrinolytic system especially D-dimers level mainly focus on normal pregnancy or normal menstrual cycle, indicating an active fibrinolytic system in a higher level under a hormonal milieu.By contrast,only few studies examined patients undergoing COH during IVF process. Available evidence suggested that D-dimers level elevated after ovarian inducing in patients suffered from ovarian hyperstimulation syndrome (OHSS).while other observation suggested a decreased D-dimers level after COH.The aim of this prospective study was to evaluate the effect of COH on plasma D-dimers level andexplore relevent factors to such change.Purpose1.To evaluate the effect of mild stimulation protocol(letrozole) and controlled ovarian hyperstimulation(COH) on plasma D-dimers level using natural cycle as a contract.2.To evaluate wheather the effect of different stimulation protocol on plasma D-dimers level were same and to explore relations between D-dimers level and estradiol and progesterone.3.To explore relations between D-dimers level and embryo implantation.Methods1.PatientsData were obtained prospectively from 2014 May to 2015 December of patients attending reproductive medicine centerof our hospital. Inclusion criteria are as follow: age<35years old (20-35 years old),with regular spontaneous menstrual cycles, normal basal steroid hormonal levels (E2:20-60pg/ml, P<lng/ml,FSH<10mIU/ml, LH<10mIU/ml), body mass index(BMI) within 18-23. Exclusion criteria are as follow:Polycystic ovary syndrome (PCOS), ectopic endometriosis, hypertension, diabetes mellitus, personal or family history of thromboembolic disease, coagulation disorder, malignant tumor, cardiovascular diseases, cerebral vascular disease, liver disease, autoimmune disease, hypothyroidism, within 30 days of infection with body temperature above 37.5℃, within 30 days after surgery or trauma, within half a year after blood transfusion, under stress the day before blood sampling; ongoing use of anticoagulants at prophylactic or therapeutic doses, history of abnormal abortion or abnormal fetal development.2.Grouping2.1 Natural cycle for frozen-thawed embryo transfer group:Patients with regular menstrual cycle received vaginal ultrasound tracking ovulation,3 days after ovulation were confirmed,2-3 embryos were transfer according to embryo quality and policy, luteal support were administered the day embryo transplant and was ongoing till blood test confirm pregnancy 2weeks after embryo transfer or otherwise.2.2 Mild stimulation for frozen-thawed embryo transfergroup:Letrozole(letrozole tablets (?),ZHEJIANG HISUN PHARMACEUTICAL CO.,LTD,Zhejiang, P.R.C) were administered for 5days starting on day5 of menstrual cycle according to weight,HMG(human menopausal gonadotropin)(human menopausal gonadotropin injection(?), Lizhu Group, Zhuhai, P.R.C)was administered depending on ovarian response, as soon as follicle reached a mean diameter of ≥18mm,5000-10000IU of hCG (Human Chorionic Gonadotrophin,Lizhu Group, Zhuhai, P.R.C) was administered,2-3 embryos were transfered 3days after confirmed ovulation, luteal support were administered the day embryo transfer and was ongoing tillblood test confirm pregnancy 2weeks after embryo transferor otherwise.2.3 Controlled ovarian hyperstimulation(COH) group:Gonadotropin Releasing Hormone agonist(GnRH-a) (triptorelin.IPSEN PHARMA BIOTECH, Signes,France) were administered on the 21st day of spontaneous menstruation.COH began on 14 days after GnRH-a administration,using r-hFSH (r-hFSH,MerckSerono, Geneva, Switzerland). The starting dose of r-hFSHwas 150 IU/day for all patients and this dose was subsequently adjusted depending on the ovarian response, as assessed by E2 levels combined with vaginal ultrasound (ALOKA SSD-3500,Tokyo,Japan). As soon as at least two leading follicles reached a mean diameter of≥ 18mm,10000 IU of hCG (Human Chorionic Gonadotrophin, Lizhu Group, Zhuhai, P.R.C) was administered and oocyte retrieval was performed by vaginal ultrasound 34-38h after hCG administration.2-3 embryos were transfered 3days after oocyte retrieval if conditions allowed, luteal support were administered the day embryo transplant and was ongoing tillblood test confirm pregnancy 2weeks after embryo transplantor otherwise.3.Labrotary method3.1 Plasma D-dimers level:Using latex agglutinate immunoturbidimetry assay under STAGO automatic analyser STA-R with kits provided by Sekisui Chemical Group, within-run precision CV<4%, betweenrun precision CV<4%.3.2Hemostasis tests were measured with anautomatic analyzer(Diatube-H; DiagnosticaStago, Asnieres, France).3.3CBC were measured with an automatic analyzer(Bayer,Leverkusen,German).3.4 FSH, LH,E2 and P concentrations were measured with Unicel Dxi800 Access (Beckman, California, the United States).4. Statistical analysis:Statistical analysis was performed using SPSS 20.0(SPSS Inc.,Chicago,IL,USA),in all analyses, P value<0.05 was considered statistically significant.4.1 Comparison withingroup:Comparison of D-dimers, hemostasis parameters and CBC between timel and time2were analyzed using wilcoxon signed rank test while comparison of same parametrics among timel,time2 and time3 were analyzed using repeated measures.Correlation analysis between plasma D-dimer level and parameters recorded were analyzed using Spearman rank correlation coefficient at time2 and time3.4.2 Comparison among groups:Comparison of matched paramers on relevant time were analyzed usingcovariance analysis with basial FSH as concomitant variable, SNK analysis were performed among groups if P value<0.05.5.Blood collection5.1 Natural cycle for frozen-thawed embryo transfergroup:Blood sample for plasma D-dimers level, fibrinogen (Fib),prothrombin time (PT), ativated partial thromboplastin time (APTT), thrombin time (TT) and a complete blood count (CBC) was drawn 2-3days after menstrual period (timel) of natural detection cycle.Another blood sample for the same tests was collected on ovulation day (time2) and 14 days after embryo transfer(time3).5.2 Mild stimulation for frozen-thawed embryo transfer group:Blood sample for plasma D-dimers level, fibrinogen (Fib),prothrombin time (PT), ativated partial thromboplastin time (APTT), thrombin time(TT) and a complete blood count(CBC)was drawn 2-3days after menstrual period (timel) preceeding treatment.Another blood sample for the same tests was collected 48h before hCG administration (time2) and 14days after embryo transfer(time3) if conditions allowed.5.3 Controlled ovarian hyperstimulation (COH) group:Blood sample for plasma D-dimers level, fibrinogen(Fib),prothrombin time(PT), ativated partial thromboplastin time (APTT), thrombin time (TT) and a complete blood count (CBC) was drawn 2-3days after menstrual period (timel) preceeding IVF-ET.Another blood sample for the same tests was collected 48h before hCG administration (time2) and 14days after embryo transfer (time3) if conditions allowed.Results1.Natural cycle for frozen-thawed embryo transfer group1.1 D-dimers level differed little between time1 and time2 (0.46(0.20,1.16)μg/ml vs 0.45 (0.20,1.09)μg/ml, Z=-0.432,P=0.666).PT and WBC increased at time2 from time1(p<0.05). Other parameters changed little from time1 to time2.For 26 patients with complete data, D-dimers level(0.46(0.20,1.16)μg/ml,0.45 (0.20,1.09)μg/ml and 0.74(0.21,1.64)μg/ml,F=9.762,P<0.001) and Fib differed among time1,time2 and time3.1.2 Correlation analysis showed factors relevant to plasma D-dimer level at time3 was serum E2 level at time2(rs=0.424, P<0.05).1.3Patients were divided into two groups based on pregnancy at time3,D-dimers level(pregnancy group(0.66(0.21,1.64) μg/ml) vs nonpregnancy group(0.82(0.56,1.22) μg/ml))and other parameters differed little between both groups.2.Mild stimulation for frozen-thawed embryo transfer group2.1 D-dimer level differed between time1 and time2(0.43(0.20,0.98)μg/ml vs0.32(0.20,0.73)μg/ml,Z=-3.626,P<0.001). APPT decreased at time2 from timel(p<0.05). WBC.PLT and PCT increased from timel to time2(p<0.05).For 18 patients with complete data, D-dimer((0.42(0.27,0.98)μg/ml), (0.30(0.20,0.73)μg/ml) and (0.80(0.41,1.56)μg/ml) respectively),Fib and WBC differed among timel,time2 and time3.2.2 Correlation analysis showed factors relevant to to plasma D-dimer level at time2 was number of mature follicles (rs=-0.436,P<0.05)whilefactors relevant to plasma D-dimer level at time3 was serum β-HCG level(rs=-0.495, P<0.05) and serum progesterone level(rs=-0.778, P<0.01).2.3 Patients were divided into two groups based on pregnancy at time3,basialD-dimers level(pregnancy group(0.31(0.27,0.65)μg/ml) vs nonpregnancy group(0.65(0.31,0.98)μg/ml)) and APTT at time3 differed between two groups,yet D-dimers level at time2 and time3and other parametrics differed little between both groups.3.Controlled ovarian hyperstimulation (COH) group3.1 D-dimers level increased from timel to time2(0.42(0.10,1.70)μg/ml, 0.56(0.20,3.14)μg/ml). PT, Fib, WBC and HGB increased at time2(p<0.01). RBC, HCT, PLT and PCTdecreased at time2(p<0.01). For 73 patients who also underwent embryo transfer at same treatment cycle, all parametrics except PDW and PT changed among timel,time2 and time3(D-dimers level at timel(0.44(0.20,1.70)μg/ml), D-dimers level at time2(0.51(0.24,1.53)μg/ml) and D-dimers level at time3(1.07 (0.20,4.38)μg/ml),respectively,F=60.944,P<0.001).3.2 Correlation analysis showed factors relevant to plasma D-dimer level at time2 was serum E2level(rs=0.160, P<0.05). Correlation analysis no factors relevant to plasma D-dimer level at time3.3.3 Patients were divided into two groups based on pregnancy at time3, all parametrics except WBC and PCT did not change significantly between both groups(D-dimers level of pregnancy group(1.14 (0.20,4.38) μg/ml) vsD-dimers level of nonpregnancy group(1.07 (0.35,1.91) μg/ml)).4.Comparison among groups4.1 Basial D-dimers level differed little among groups(Natural cycle for frozen-thawed embryo transfer group(0.46(0.20,1.16) μg/ml),Mild stimulation for frozen-thawed embryo transfer group(0.43(0.20,0.98) μg/ml),Controlled ovarian hyperstimulation (COH) group(0.42(0.10,1.70) μg/ml)),yet difference among groups showed significantly at time2 and time3(P<0.01). APTT,Fib and TT differed among groups at time2(P<0.05) while other parameters differed ltiile among groups(P>0.05).4.2 WBC of COH group differed from other groups at time2 and time3(P<0.01).HGB,PLT,HCT and PCT of letrozole group differed from COH groups at time2(P<0.01).Other parameters changed little among groups at the same time(P>0.05).Conclusion1.Present results suggest that D-dimers level increased from time1 to time2 in natural cycle for embryo transfergroup.While in mild stimulation group D-dimers level decreased in time2 and raised in time3.Yet in COH group D-dimers level increased subsequently among 3times.Changing pattern of D-dimers level differed among three groups using natural cycle group as contract group,such pattern may be related to changing sex hormone level among different protocol.2. Present results suggest that D-dimers level at time3 was related to E2 level at time2 in natural cycle group.In mild stimulation group, D-dimers level at time2 was negatively related to number of mature follies at time2 while D-dimers level at time3 was negatively related to serum β-HCG and P.D-dimers level was positively related to serum E2 level at time2 in COH group.Factors related to D-dimers level remained unspecific among different protocol,yet all factors resulted from ovulation inducing drug or luteal support medicine.3. Patients were devided into two groups based on pregnancy at time3,present results suggest that D-dimers level changed little among time1,time2 and time3 in natural cycle group and COH group.In mild stimulation group,basial D-dimers level in nonpregnancy group was higher than in pregnancy group while such difference disappeared at time2 and time3.Although present results failed to connect D-dimer level to pregnancy, embryo implantation may be influenced by lots of factors which present study failed to balance,future study balanced such factors are in need.4. Through horizontal comparison to explore change of D-dimers level among three groups, D-dimers level changed little in natural cycle group and mild stimulation group while changed dramatically in COH group.such change was related to serum E2 level,indicating dramatic changes of sex hormones may induce an elevation in fibrinolytic potential,resulting in elevated D-dimer level.5.Though responsible mechanism remains unclear,future larger studies are required to clarify these issues, when it comes to ovulation induction,thrombotic complication such as OHSS must beware of. |
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