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    Serum β-hCG level on day 7 of frozen-thawed embryo transfer:association with the clinical pregnancy outcomes in artificial cycles

    2022-08-17 08:26:30NaSunShuYiDongPingPingSunHuaGangMa
    Clinical Research Communications 2022年3期

    Na Sun ,Shu-Yi Dong ,Ping-Ping Sun ,Hua-Gang Ma*

    1Center of Reproductive Medicine,Weifang People's Hospital,Weifang 261000,China.2Reproductive Health Department,Gaomi Maternity and Child Care Hospital,Weifang Gaomi 261500,China.

    Abstract Objective: The relationship between serum beta human chorionic gonadotropin (β-hCG)levels of patients (7 days after the transplantation of frozen-thawed embryos) and the pregnancy outcomes was investigated.Methods:This study was designed as a retrospective clinical trial of 366 women who underwent frozen-thawed embryo transfers (FETs) in artificial cycles.Patients were divided into three groups:clinical pregnancy group,biochemical pregnancy group,and non-pregnant group according to their pregnancy outcomes.Serum β-hCG levels were tested on day 4,7,9,11 and 14 after FET.Results: In the clinical pregnancy group,the serum β-hCG levels after 7-day post-transplantation were significantly elevated (16.20 IU/L vs.3.07 vs.0.1 IU/L;P <0.05) compared with the other two groups.Furthermore,it was found that Area Under Curve (AUC=0.96) was significant with cut-off value higher than 4.26 IU/L (sensitivity=92.3%,specificity=90.2%) to predict the clinical pregnancy outcomes in the receiver operating characteristic (ROC)analysis of β-hCG concentrations on day 7 of post-transplantation.Conclusion: Our results suggested that the elevated serum β-hCG levels on day 7 of post-transplantation could predict the positive clinical pregnancy outcomes in artificial FET cycles.

    Keywords: frozen-thawed embryo transfer;pregnancy outcomes;artificial cycle;human chorionic gonadotrophin;infertility

    Background

    The general aim of assisted reproductive technology (ART) is to achieve pregnancy using techniques such asin vitrofertilization and eventually to have the delivery of a healthy baby.Frozen-thawed embryo transfer (FET) has become an essential part ofin vitrofertilization (IVF),which has been reported to elevate implantation and ongoing pregnancy rates freeze-all approach relative to fresh transfer cycles[1].The benefits of FET could be explained by avoiding a negative effect of ovarian hyperstimulation on the endometrium and thereby improve embryo implantation[2].Early-stage(within 7 days)prognosis of pregnancy status is of critical importance.Specifically,the identification of a prognostic test that can be used to reflect the pregnancy status at day 7 post-FET would be helpful not only to alleviate the anxiety of patients undergoing assisted reproduction technology but also promote more effective doctor-patient consultation processes.Currently in the ART field,it is known that beta human chorionic gonadotropin (β-hCG) can be measured in the serum as a specific and sensitive marker to predict early pregnancy[3,4].The serum beta-hCG levels were usually detected on 12-14 days after embryo transfer [5-7].Study found a predictive value of pregnancy outcome with serum hCG drawn 7 days after D3 embryos transfer in IVF .In this study,we explored serum β-hCG levels at the 7th-day after FET.It may be an effective prediction for clinical pregnancy in FET.

    Materials and methods

    Patient recruitment

    The study was designed as a retrospective clinical trial approved by the Medical Ethics Board of Weifang People’s Hospital (2018091302).All the participants signed informed consent form.A total of 366 patients undergoing FET treatment were enrolled at the Center of Reproductive Medicine,Weifang People’s Hospital,Weifang(Shandong,China).Inclusion criteria:a.hormone replace treatment(HRT) was used for endometrial preparation;b.patients were transferred with D3 high quality cleavage-stage embryos;c.the same luteal support.The exclusion criteria were ovarian cysts,hydrosalpinx,uterine cavity,and endometrial lesions.Patients with ectopic pregnancy post FET were excluded .

    Endometrial preparation and luteal support

    8 mg estradiol (Femostone,Abbott Biologicals B.V.) was administered from day 2-3 of menstruation.When the endometrial thickness reached 7 mm (as detected by transvaginal sonography),endometrium was further transformed with 60 mg progesterone (20 mg,Zhejiang Xianju Pharmaceutical Co.,China) per day for 3 days.No more than two D3 embryos were transferred.Luteal support included progesterone and Femostone (2 mg estradiol plus 10 mg dydrogesterone).

    Detection of serum hormones

    Serum estradiol (E2),progesterone (P) and β-hCG levels were monitored in all patients on the day 4,7,9,11 and 14 after transplantation by chemiluminescence (Roche,Germany).Venous blood samples were collected before 9 a.m.

    Grouping of patients

    Patients were divided into three groups:group A was the clinically pregnant group,group B was the biochemically pregnant group,and group C was the non-pregnant group.According to the outcomes of clinical pregnancy they were further divided into a normal pregnancy subgroup (including a single continued pregnancy subgroup and a twins continued pregnancy subgroup) and an abnormal pregnancy subgroup (including an early spontaneous abortion).Clinical pregnancy refers to the appearance of the gestational sac by ultrasound after FET.Biochemical pregnancy is defined as the detection of hCG in blood or urine without subsequent clinical signs of pregnancy.Early spontaneous abortion means pregnancy loss after the formation of intra-uterine sac visualized by pelvic ultrasonography before 12 weeks gestation.Single or twins continued pregnancies are defined as continued pregnancy over 12 weeks.

    Statistical analysis

    SPSS 20.0 was used for statistical analysis.For group comparisons,we used a parametric test,analysis of variance (ANOVA),and non-parametric tests (Kruskal-Wallis test,Chi-square test).Bonferroni adjustment for multiple comparisons was used when indicated.All values were presented as the mean ± standard deviation or percentage.P <0.05 was considered statistically significant.The area under the receiver operating characteristic (ROC) curve (AUC) was calculated to assess the accuracy of utilizing the β-hCG concentrations to predict the pregnancy probability.

    Results

    Elevated level of the maternal age in the non-pregnant group compared with the pregnant groups

    The average age in the non-pregnant group was significantly higher than the two groups (31.96 vs.33.53 vs.37.24,P< 0.05).Interestingly,there was no significant difference in age found between the clinical and biochemical pregnancy groups (31.96 vs.33.53,P=0.175).Among the subgroups,the age in twins continued pregnancy was younger than other two subgroups(29.76 vs.31.67 vs.34.29,P<0.05).No difference was found between single continued pregnancy and early spontaneous abortion subgroups (31.67 vs.34.29,P=0.087).The infertility duration and body mass index were similar in three groups (2.74 vs.3.02 vs.2.41,P=0.165;23.93 vs.24.48 vs.24.26,P=0.552) and subgroups (2.44 vs.2.85 vs.2.87,P=0.553;24.50 vs.24.08 vs.22.87,P=0.100).

    Serum E2 and P showed no difference among three groups

    There were no differences among three groups in serum E2 and P in the end of proliferation period (E2:1798 vs.1527 vs.1764 pmol/L,P=0.689),on the transfer day (E2:1584 vs.1265 vs.1558 pmol/L,P=0.351;P:56.96 vs.51.36 vs.57.73 nmol/L,P=0.34) and on the day 14 after embryo transfer(E2:1770 vs.1331 vs.1345 pmol/L,P=0.055;P:114.91 vs.96.07 vs.114.06 pmol/L,P=0.066) (Table 1).

    Serum β-hCG levels significantly increased on day 7 of clinical pregnancy group compared with group B&C

    Serum β-hCG levels showed significantly different on day 7,9,11 and 14 after FET within three groups (Table 2).The β-hCG levels were 16.20 vs.3.07 vs.0.1 IU/L (P <0.05) for the three groups on the 7th day after FET (Table 2).We used ROC curve to further analyze the diagnostic accuracy of serum β-hCG after FET (Table 3).The AUC achieved was significant between clinical pregnancy and non-pregnancy on day 7,9,11 and 14 (Figure 1).β-hCG levels measured 7 days after FET were the earliest points to predict clinical pregnancy.When β-hCG ≥4.26 IU/L was used to distinguish clinical pregnancy and non-pregnancy,the AUC was 0.961 with a sensitivity of 92% and a specificity of 90%.Further,the same significant differences appeared in β-hCG on day 9,11 and 14.

    Figure 1 ROC curve of β-hCG on day 4,7,9,11 and 14 to predict clinical pregnancy after FET

    Serum β-hCG levels significantly increased on day 7,9,11 and 14 of twins continued pregnancy subgroup compared with two subgroups (a singleton continued pregnancy subgroup and an early spontaneous abortion subgroup)

    Table 4 showed significant differences of β-hCG levels from day 7 to day 14 post FET among three subgroups except day 9.On 9th day,serum β-hCG level in twins continued pregnancy subgroup was higher than other two subgroups.No significant difference was found in the remaining subgroups.

    Table 1 Changes in hormone levels after FET

    Table 2 Changes in hormone levels after FET

    Table 3 Cutoff points and predictive efficacy of day-4,7,9,11,14 serum β-hCG for FET pregnancy outcomes

    Table 4 Changes in β-hCG levels after FET among subgroups

    Discussion and conclusions

    Human chorionic gonadotropin is a hormone comprising an α-subunit and a β-subunit which are held together by hydrophobic ionic linkages [8].The β-subunit of hCG is structurally highly specific.Therefore,as a key embryonic signal of hCG,detection of β-hCG in blood and urine samples has been a sensitive method for early pregnancy diagnosis [9].The trophoblast cells began to secrete trace amounts of hCG on 6th day after fertilization and β-hCG can be detected from maternal serum 10 days after fertilization [10].In exploring the relationship between β-hCG and pregnancy outcomes,as well as avoiding the residual effect of HCG trigger,the serum β-hCG levels were usually investigated on the 12 to 14th day after embryo transplantation [5-7,11].Findings of Shapiro suggested embryonic implantation and developmental fate are largely determined by 5 days after blastocyst transfer and that very early serum HCG measurements may be useful markers of IVF outcomes [12].H.Kim study found the hCG values ranged from 13 to 15 days after oocyte retrieval according to fertilization methods were not significantly different both in singleton and twin pregnancies [13].Wang concluded hCG cutoff value of 2.5 IU/L is predictive of pregnancy 7 days after D3 embryos transfer in IVF [14].In this study we want to investigate whether a similar conclusion of earlier β-hCG test (7 days post-FET) could be achieved to indicate clinical pregnancy in artificial cycle of FET,in which no HCG was used.We divided enrolled patients into three groups:clinical pregnancy group,biochemical pregnancy group and non-pregnancy group.All the patients were given identical adequate amount of luteal support.Serum β-hCG levels significantly increased in the clinical pregnancy group on the 7th day after FET.It is well known that serum β-hCG levels during early pregnancy is determined by the number and the development extent of the villi.The 7th day after transplantation is equivalent to 10th day afterin vivofertilization.Therefore,serum β-hCG was detected on 7th day from pregnant women understandably,consistent with previous research[14].In this study,we collected patients,blood frequently,one is for observing whether the luteal support was sufficient and the other was to track the trends of β-hCG.Serum β-hCG levels were expected to have normal growth trends in the following days in the clinical pregnancy group.Our secondary objective was to determine whether β-hCG levels on 7th day indicated continued pregnancy and the relationship between singleton and multiple pregnancies.Póvoa Ana concluded β-hCG at 13 d after embryo transfer might predict outcomes in clinical singleton and twin pregnancies following IVF.A concentration of β-hCG >386 IU/L had a 91% chance of a live birth,whereas a level >768 IU/L associating with 81% chance of live twin birth [15].In our found,serum β-hCG showed a significant difference 7 day after FET in twins continued pregnancy than the other subgroups,which could be explained by the higher villi activity in multiple pregnancies.

    In this study,groups of biochemical pregnancy and non-pregnancy indicated the difference in age compared with clinical pregnancy group,in agreement with the former studies that age was the important predictive factor contributing to pregnancy outcomes[16-18],due to the associated gradual depletion of the follicle pool and lower quality of oocytes in the aging population [19].

    In conclusion,the increase of serum β-hCG levels on the 7th day after FET can provide significant insights into predicting clinical pregnancy in a more effective manner.This may provide potential guidance for the implementation of the next treatment plan for FET patients.

    We obtained clinical data from hundreds of patients,concluding serum β-hCG on the 7th day after FET was meaningful.Considering over-frequent blood drawing is troubling to patients,we will reduce frequency of blood drawing and expand the sample size to explore the relationship between the early serum β-hCG and live birth rate.Ectopic pregnancy will be also included and analyzed.

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