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Journal of Reproduction & Contraception doi: 10.7669/j.issn.1001-7844.2013.03.0151

2013 Sep.; 24(3):151-158 E-mail: randc_journal@163.com

Reducing the Trigger Dose of Human Chorionic Gonadotrophin

Does Not Affect Final Oocyte Maturation and Subsequently

Pregnancy Outcome of Frozen-thawed Embryo Transfer

Yun WANG*, Yong-lun FU*, Shao-feng CAO, Qiu-ju CHEN, Song-guo XUE, Qi-feng LYU,

Yan-ping KUANGDepartment of Assisted Reproductive, Shanghai Ninth People’s Hospital, Shanghai Jiao Tong University School

of Medicine, Shanghai 200011, China

Objective To compare the efficacy of human chorionic gonadotrophin (hCG) atreduced doses of 2 000 IU and 3 000 IU for moderate or high responders with thedose of 5 000 IU in term of inducing final oocyte maturation for IVF/ICSI and thesubsequent pregnancy outcome in frozen-thawed embryo transfer (FET).Methods In the retrospective cohort study, 2 166 patients undergoing IVF/ICSI withmoderate or high response were recruited and classified into three groups accordingto the trigger dose of hCG: 2 000 IU (group A, n=722), 3 000 IU (group B, n=722)and 5 000 IU (group C, n=722). The main outcome was the proportion of matureoocytes retrieved, fertilization rates, clinical pregnancy rates, cumulative pregnancyrates and incidence of ovarian hyperstimulation syndrome (OHSS).Results No evidence of statistically difference was found in the proportion of matureoocytes retrieved (89.92%, 91.40%, 90.20%, respectively) and fertilization rate(79.8%, 80.07%, 80.51%, respectively) among groups A, B and C. Serum E2 level onthe day of hCG injection, the number of mature oocytes retrieved and good-qualityembryos in group A were significantly higher than those in group B and group C.Clinical pregnancy rates per transfer cycle (45.95%, 43.97% and 44.25%), ongoingpregnancy rates (43.17%, 40.91% and 42.53%), implantation rates (30.74%, 27.78%and 29.86%) and cumulative pregnancy rates per patient (58.31%, 53.6% and 54.85%)

This study was supported by National Natural Science Foundation of China (No. 31071275) and Natural Science Foundation of Shanghai of China (No. 09411962900)Corresponding author: Qi-feng LYU; Tel:+86-21-23271699*5538; Fax: +86-21-53078108; E-mail: lyuqifeng@126.com Yan-ping KUANG; E-mail: Kuangyanp@126.com*: These two authors contributed equally to this paper

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were comparable among groups A, B and C. The incidence of OHSS in groups A, B andC (0%, 0.14% and 0.28%) was low, with no significant difference.Conclusion A reduced hCG dose of 2 000 IU for moderate or high responders leadsto similar efficacy compared with a dose of 5 000 IU in inducing oocyte maturationwithout adversely affecting the pregnancy outcome meanwhile eliminating the risk ofOHSS.

Key words: human chorionic gonadotropin (hCG); controlled ovarian hyperstimulation (COH); ovarian hyperstimulation syndrome (OHSS); frozen-thawed embryo transfer (FET); cumulative pregnancy rate (CPR)

The use of exogenous human chorionic gonadotropin (hCG) to mimic the ovulation

inducing LH surge is of great importance in the field of reproductive medicine. Due to its

high LH receptor affinity and low disappearance rate, this exogenous hCG causes a

nonphysiological high and long lasting stimulation of the luteal body[1,2]. The direct consequence

of an exaggerated follicular response to controlled ovarian stimulation (COH) in the

presence of hCG is the initiation of ovarian hyperstimulation syndrome (OHSS)[3]. In most

cases, OHSS is self-limiting and resolves spontaneously within several days, however,

moderate or severe OHSS can cause significantly morbidity and be fatal in its critical stage[4].

There is no existing data on the optimal dose requirements of hCG for final oocyte

maturation. Under the examination of microscope, the occurrence of the first polar body is a

hallmark of oocyte maturation. The maturation of ooplasm plays an important role in the

process of fertilization and early development of embryos. Sperm adherence and penetration

are highly related to the maturation of the ooplasm[5]. Doses of hCG as low as 3 300 IU[6]

have been shown to effectively trigger oocyte maturation in assisted reproductive technology

(ART) without adversely affecting cycle outcome; 2 000 IU was ineffective[7]. Doses of

hCG as low as 2 500 IU have been shown to be effective in patients with PCOS[8]. The

reducing dose of hCG was proved to be able to prevent the development of OHSS[8] or

unable to prevent OHSS in high-risk women[6].

In recent years, we attempted to identify the optimal effective doses of hCG trigger

and found that adequate oocyte maturation could be obtained with the dose of 2 000 IU

or 3 000 IU when administered to moderate or high responders. We used a strategy of lower

dose of hCG (2 000 IU or 3 000 IU) in combination with cryopreservation of all embryos for

moderate and high responders to prevent the developing of OHSS. Therefore, the retrospective

study was aimed to assess whether the lower trigger doses of hCG (2 000 IU or 3 000 IU)

equally induce final oocyte maturation and subsequent pregnancy outcome in frozen-thawed

embryo transfer (FET) cycles compared with the dose of 5 000 IU.

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Materials & Methods

Study design and participants

This was a retrospectively observational study of patients undergoing IVF/ICSI practice.

The consecutive 2 166 patients who underwent induced ovulation using long protocol and

subsequent FETs from February 1, 2011 to July 31, 2012 were collected and grouped

according to the dose of hCG 2 000 IU (group A), 3 000 IU (group B) and 5 000 IU (group

C) for triggering ovulation.

The inclusion criteria was: 1) basal follicle-stimulating hormone (bFSH)<10 mIU/ml

with body mass index (BMI) 25 kg/m2; 2) serum estradiol (E2) 2 000 pg/ml and no less

than 10 follicles with 10 mm in diameter on the day of hCG administration; 3) adequate

sperm for IVF or ICSI.

Study process

Ovarian stimulation and embryo culture

Ovarian stimulation was facilitated by the long protocol with 50 μg of Decapeptyl (Ferring

International Center SA, Germany) given SC starting on day 21 of the preceding cycle. hMG

(Anhui Fengyuan Pharmaceutical Co., China) was given at a dose of 150 225 IU/d from

day 3 of menstruation and Decapeptyl was continued until the day of hCG administration.

The dose of hMG was adjusted according to oestradiol concentrations and ovarian response.

hCG (Guangdong Lizhu Pharmaceutical Co., China) was used to trigger ovulation when at

least one follicle was more than 20 mm or two follicles were more than 18 mm in diameter.

Oocyte retrieval was carried out 36 37 h after the hCG triggering. Fertilization was

carried out in-vitro either by conventional insemination or ICSI depending on the semen

parameters and previous fertilization history. Embryos were examined for the number/

regularity of blastomeres and the degree of fragmentation, and graded according to the

criteria of Cummins[9]. OHSS cases were classified according to previously published

classification system[10].

All good-quality embryos were frozen on the third day of oocyte retrieval. The non-

good-quality embryos were thus placed for further extended culture until blastocyst stage

and only good morphology blastocysts were frozen. Procedure of freezing-thawing of cleavage-

stage embryos and blastocysts has been described previously[11]. Thawed embryos were

classified as either fully intact (100% cells survived), partially damaged ( 50% cells survived)

or degenerated (<50% cells survived)[12]. Only intact and partially damaged embryos were

considered as survival and would be transferred.

Endometrial preparation and FET

Patients were prepared for FET with daily oral administration of 8 mg estradiol valerate

(Abott Biological B.V, Netherland) started from day 3 to attain the criteria of endometrial

thickness 8 mm and tripleline pattern on ultrasound scans. At that time, patients were

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given 0.4 g progesterone (Laboratoires Besins-Iscovesco, France) intravaginal daily and

embryo transfer was performed 3 d later under abdominal ultrasound guidance. Oral

estradiol and progesterone were continued until documentation of fetal heart activity by

ultrasonography[13].

Outcome measures

The primary outcome measure was the proportion of mature oocytes (metaphase II

oocyte) retrieved per patient following hCG administration. Percentage of oocytes

retrieved=the number of oocytes retrieved/the number of follicles punctured 100%,

percentage of mature oocytes retrieved=the number of mature oocytes retrieved/the

number of oocytes retrieved 100%, fertilization rate=the number of fertilized oocytes/the

number of mature oocytes retrieved 100%. The secondary outcome included the fertili-

zation rate, the clinical pregnancy rate per transfer cycle and the cumulative pregnancy rate

per patient. Clinical pregnancy was considered as the presence of a gestational sac with

fetal heart activity, as assessed by ultrasound at 7 weeks of gestation. Ongoing pregnancy

was defined as pregnancy proceeding beyond the 12th gestational week. Cumulative

pregnancy was defined as the total number of pregnancy using multiple FETs.

Statistical analysis

Data were expressed as mean SD (x s) or percentage (%). Statistical analysis

was performed by SPSS16.0. χ2 test was used for categorical comparisons or Fisher’s

exact tests as appropriate. One-way ANOVA was used for continuous variables. The

significant difference was considered at P value less than 0.05.

Results

A total of 2 166 patients had completed the oocyte retrieval cycles and were analyzed.

A total of 2 852 FET cycles were performed during the research period. There were no

significant differences regarding demographic characteristics among the three groups

(Table 1).

The clinical and embryological characteristics of the population are demonstrated in

Table 2. The duration of stimulation was similar among the three groups (P>0.05). Total dose

of gonadotropin consumption in group A was significantly lower than that in group C (P<0.05).

Serum E2 level on the day of hCG administration, the number of oocytes retrieved, the

number of mature oocytes in group A were higher than those in group B meanwhile those

indexes in group B were higher than those in group C (P<0.05). No significant difference

was found in the proportion of oocytes retrieved, the mature oocyte rate and the fertilization

rate among the three groups (P>0.05). The embryo cryosurvival rate was about 98%.

There was no significant difference in the number of embryos transferred and thepregnancy outcome of the FET cycles (Table 3). The clinical pregnancy rate per transfer was

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Table 1 Patients characteristics (x s or %)

Parameter Group A Group B Group C

nAge (year)

BMI (kg/m2)

Day 3 FSH (IU/ml)

Duration of infertility (a)

Cause of infertility (%)

Tubal factor

Male factor

Ovulatory factor

Endometriosis

Unexplained

722

30.6 3.3

21.66 2.25

6.92 1.91

4.5 2.8

69.4 (501/722)

14.5 (105/722)

9.0 (65/722)

2.5 (18/722)

4.6 (33/722)

722

30.4 3.5

21.59 3.08

6.89 1.73

4.0 2.7

67.5 (487/722)

16.2 (117/722)

8.9 (64/722)

3.0 (22/722)

4.4 (32/722)

722

29.9 2.8

21.34 2.95

7.11 1.58

4.3 2.9

66.6 (481/722)

14.7 (106/722)

9.4 (68/722)

3.3 (24/722)

6.0 (43/722)

Table 3 Treatment outcomes

Parameter Group A Group B Group CClinical PR per transfer (%)

Ongoing PR per transfer (%)

Multiple pregnancy rate (%)

Implantation rate (%)

Cumulative PR per patient (%)

PR: pregnancy rate

45.95 (448/975)

43.17 (421/975)

27.08 (114/421)

30.74 (535/1 740)

58.31 (421/722)

43.97 (416/946)

40.91 (387/946)

28.94 (112/387)

27.78 (499/1 796)

53.60 (387/722)

44.25 (412/931)

42.53 (396/931)

26.51 (105/396)

29.86 (501/1 678)

54.85 (396/722)

Table 2 Cycle characteristics (x s or %)

Parameter Group A Group B Group Cn

Duration of stimulation (d)

Total Gn consumption (IU)

E2 on the day of hCG injection (pg/ml)

No. of oocytes retrieved (n)

Percentage of oocytes retrieved (%)

No. of mature oocytes retrieved (n)

Percentage of mature oocytes (%)

ICSI performance (%)

Fertilization rate (%)

No. of embryos vitrified (n)

No. of embryos warmed per transfer cycle (n)

Patients without vitrified embryos (%)

Rate of moderate and severe OHSS (%)

*: P<0.05, compared with group C

#: P<0.05, compared with group B

722

10.3 2.4

1 950.13 624.93*

3 841.29 1 367.9*#

15.19 8.39*#

69.70 19.00

12.8 7.6*#

89.92 17.63

17.04 (123/722)

79.80 20.12

6.0 4.2*#

1.8 0.5

3.19 (23/722)

0.00 (0/722)

722

10.6 2.9

2 048.89 668.77

3 027.29 1 595.14*

13.96 7.97*

68.87 21.91

11.4 6.9*

91.40 14.23

15.93 (115/722)

80.07 17.68

5.5 3.7*

1.9 0.3

2.90 (21/722)

0.14 (1/722)

722

10.5 2.5

2 105.77 715.65

2 845.86 1 027.58

10.94 6.47

71.00 18.19

8.0 5.9

90.20 10.36

15.24 (110/722)

80.51 16.19

4.3 3.0

1.8 0.2

3.60 (26/722)

0.28 (2/722)

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45.95% in group A, 43.97% in group B and 44.25% in group C, with no significant difference(P>0.05). The cumulative pregnancy rates per patient were 58.31% in group A, 53.60% ingroup B and 54.85% in group C, without significant differences (P>0.05).

The incidence of moderate and severe OHSS is low in the study and no difference wasfound among groups A, B and C (0%, 0.14%, 0.28%, respectively, P>0.05). Only 3 cases werediagnosed as early-onset moderate OHSS, including one case being hospitalized in group C.No case of OHSS was found in group A. No incidence of late-onset OHSS or severe OHSSoccured in the study.

Discussion

The hCG-imitated LH surge induces the dispersion of granulose cells around the oocytes.Thus, the oocytes are easy to be retrieved. The number of retrieved oocytes is closelycorrelated with the dose of hCG. This study demonstrated statistical and clinical equivalenceof three doses of urinary hCG in moderate or high responders for induction of final follicularmaturation in patients undergoing IVF. The heavy of this study is its large scale. Our studyhas adequate power for achieving our primary and secondary outcome measures such aspregnancy rates and incidence of OHSS.

hCG has been used in various IVF treatment protocols to induce final maturation butthere is as yet no agreement on the minimum dose required. Studies in nonhuman primateshave shown that hCG doses 3- to 10-fold lower than standard doses have been sufficient tore-initiate meiosis and induce luteinization of granulosa cells[14]. Abdalla et al.[7] reported thatthe oocyte recovery rate in patients who received 2 000 IU hCG was lower compared withpatients who received either 5 000 IU or 10 000 IU hCG, but the study was performed twodecades ago and it was possible to make room for the development of ART. The laterstudies in an effort to reduce the risk of OHSS in high responder patients using 2 500 IU[8],3 300 IU[6], with the promising pregnancy outcome. Up to our known, it is the first large scalestudy to establish a hCG dose of 2 000 IU as an effective dose for triggering ovulation inIVF/ICSI cycles.

On the other hand, the common dose of 5 000 IU or 10 000 IU hCG is proved that itcould not only induce ovulation, but also mean a long lasting stimulation of the luteal body forthe next days. In this study, whether the lower dose of hCG plays a reduced role in lutealsupport could not be explored due to cancel all fresh embryo transfer cycles. Lower dose ofhCG may affect the maturity of ooplasm, the quality of embryo and the development of earlyembryo, so we generalized the research results with caution that patients may have differentthresholds for follicular response to hCG and the recommended minimum dose of hCGshould be at least 2 000 IU.

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Another highlight of the study is the low incidence of OHSS. Moderate to severe OHSShas been calculated to occur in 0.2% to 2% of all ovarian stimulation cycle[15]. Triggeringwith 5 000 IU or 10 000 IU appears to be effective as far as oocyte recovery is concerned.However, both dosing schedules may lead to severe OHSS[16]. With its relatively long serumhalf-life, hCG has certain detrimental side effects. The persistence of hCG in the serumafter administration can lead to delayed ovulation from smaller follicles, development ofmultiple corpora lutea, sustained luteotropic effects and an increased risk of OHSS. Therefore,reducing the dose of hCG regimen can be useful in decreasing the risk of OHSS in moderateor high ovarian responders.

Cryopreserving of all embryos with transfer in a subsequent cycle protects a patientfrom late onset OHSS. FET also avoids the negative effects of embryo-endometriumasynchrony in fresh stimulation cycles and improves the pregnancy outcome[13,17]. Thus, itprovides many clinical benefits, including increasing the cumulative pregnancy rate andreducing the risk of OHSS.

In conclusion, the study demonstrated that an hCG dose of 2 000 IU for moderate orhigh responders led to similar efficacy compared with a dose of 5 000 IU in inducing oocytematuration without adversely affecting the pregnancy outcome. At the same time, the risk ofOHSS in a high responder population is dramatic eliminated, therefore, the strategy ofreducing trigger dose of hCG and cryopreserving all embryos for later transfer isrecommended for the patients with high-risk OHSS in IVF treatment.

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(Received on April 25, 2013)