Journal of Reproduction & Contraception

doi: 10.7669/j.issn.1001-7844.2014.02.0089

2014 Jun.; 25(2):89-96

E-mail: randc_journal@163.com

Revised Super-long Down-regulation Protocol Improves

the Outcome of Infertile Patients with Repeated Implantation Failure in IVF/ICSI-ET
Min-feng SHI1*, Tie ZHOU2*, Ying WANG1, Guang-hua CHEN2, Ming ZHU1, Jun OU1 ,
Hui DU1, Tao-fei YAN1, Di SONG1, Ju-fen ZHENG1, Hui-qin ZHANG1, Ning HUI1 ,
Ying-hao SUN2
1. Reproductive Centre, Changhai Hospital, the Second Military Medical University, Shanghai 200433, China
2. Department of Urology, Changhai Hospital, the Second Military Medical University, Shanghai 200433,
China

Objective To investigate the outcome of revised super-long down-regulation protocol

(RSDP) for in vitro fertilization / intracytoplasmic sperm injection-embryo transfer
(IVF/ICSI-ET) in the special infertile patients with repeated implantation failure (RIF).
Methods Patients with RIF were divided into RSDP group and routine long downregulation protocol (RLDP) group. In RSDP group, gonadotropin releasing hormone
agonist (GnRHa) was injected intramuscularly by 2.5 mg in mid-luteal phase for the first
time and 1.25 mg after 28 d; gonadotropin (Gn) was started 14 d later after the second
GnRHa dose. IVF/ICSI-ET was performed according to the routine procedure. The
clinical outcomes of RSDP group were compared with those of RLDP group.
Results
In RSDP group, the number of retrieved oocytes and valid embryos was
significantly lower (P<0.05); there were no significant differences about fertilization
rate (P>0.05); both good-quality embryo rate and implantation rate were significantly
increased (P<0.005); clinical pregnancy rate was obviously improved (P<0.05), as
compared with RLDP group.
Conclusion

RSDP can improve the IVF outcomes significantly in RIF patients.

Key words: implantation failure; revised super-long down-regulation protocol (RSDP);

routine long protocol; in vitro fertilization (IVF)

Corresponding author: Ying-hao SUN; Tel: +86-21-31161718; E-mail: sunyh@medmail.com.cn

*: These authors contribute equally to this work
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Now, routine long down-regulation protocol (RLDP) and other controlled ovarian
hyperstimulation (COH) protocol have been commonly applied to infertile patients for
treatment of in vitro fertilization/intracytoplasmic sperm injection-embryo transfer (IVF/
ICSI-ET) and got high pregnancy rate, but there were still some special patients with
repeated implantation failure (RIF). RIF was determined when transferred embryos failed
to implant after several IVF treatment attempts[1]. However, there were no formal criteria
defining the number of failed cycles or the total number of embryos transferred in IVF
attempts. The incidence rate of RIF was about 10%. As a highly coordinated event,
successful embryo implantation mainly depends on well-functioning endometrium as well as
excellent quality embryos. How to improve the clinical pregnancy rates of these special
patients was one of the tough problems for IVF. Since good COH protocol could result in
multiple synchronized developed follicles and high quality mature oocytes, and improve
fertilization rate and pregnancy rate, we applied revised super-long down-regulation protocol
(RSDP) to these patients in our centre, and IVF outcomes were compared between RSDP
and the control group of RLDP. To our knowledge, this is the first comparison of these two
protocols in RIF population.

Materials & Methods

General data
In our centre, RIF was defined as the absence of a gestational sac on ultrasound
at 5 weeks post-embryo transfer after 3 embryo transfers with high-quality embryos or
the transfer of 10 embryos in multiple transfers[2]. According to this criterion, a total of 112
patients with RIF in our centre from January 2010 to November 2013, were divided into
RSDP group and RLDP group. Hysteroscopy was routinely performed in all of these RIF
patients to assess the uterine cavity and have treatment accordingly. The age, reasons and
duration of infertility, basal endocine parameters and the means of assisted conception were
compared between the two groups.
Methods
Revised super-long down-regulation protocol
GnRHa (long-acting triptorelin, Beaufour Ipsen Pharmaceutical Co. Ltd) 2.5 mg was
injected intramuscularly 7 d after ovulation or in the 16th day of the oral contraceptive cycle
with Diane 35 (Ethinylestradiol and Cyproterone Acetate Tablets, Schering GmbH & Co.
Produktions KG), and repeated 28 d later with the dose of 1.25 mg. Follicle stimulating
hormone (FSH, Gonal-F, Serono) was used for follicle development 14 d later.
Routine long down-regulation protocol
GnRHa 1.25 mg was injected intramuscularly once at mid-luteal phase for downregulation or in the 16th day of the oral contraceptive cycle with Diane 35. FSH was used for
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COH 14 d later.
Evaluation of the follicle development
Ultrasonograph combined with serum hormone measurement was used for evaluation
of the follicle development. Recombinant human chorionic gonadotropin (Merck Serono)
0.25 mg was injected to promote the maturation of the oocytes when at least one of the
dominant follicle reached 18 mm in diameter or two with 17 mm in diameter. Oocytes were
retrieved after 34-36 h. The culture of oocytes and embryos, fertilization, ICSI, and embryo
transfer were performed according to the standard protocols. The embryos were transferred at 4-8 cells and 1-3 embryos were transferred. Clinical pregnancy was confirmed by
elevated serum hCG level and pregnancy sac identified by ultrasonography.
Statistical analysis
Data were expressed as mean standard deviation (x- s), or percentage (%). SPSS
10.0 software (SPSS Inc., Chicago, IL, USA) was used for statistical analysis. T test and
chi-square were used respectively. P<0.05 was deemed statistically significant.

Results
The characteristics of patients are shown in Table 1. Ten cycles were cancelled in
RSDP group and RLDP group respectively for failed oocytes retrieval, unfertilization, or
cleavage failure. The reasons for cycle cancellation are shown in Table 2. The ET cycles in
RSDP group and RLDP group were 42 and 50, respectively. As for category, reasons, and
duration of infertility, body mass index (BMI), basal E2 and the ratio of IVF/ICSI cycles,
there were no significant differences between the two groups (P>0.05). But the patients
Table 1 Characteristics of the patients
Item
Primary infertile (n)

RSDP group
30

RLDP group
36

P
>0.05

Secondary infertile (n)

22

24

>0.05

Infertile duration (year)

5.8 3.0

4.8 2.9

>0.05

Age (year)

33.8 5.1

29.6 4.2

<0.05

BMI (kg/m2)

22.0 3.2

22.3 3.5

>0.05

Basal E2 (pg/ml)

56.8 30.2

57.0 32.6

>0.05

8.0 1.8

6.8 2.6

<0.05

Basel FSH (IU/ml)

Etiology

>0.05

Male factor (%)

9.5

20.0

Tubal factor (%)

57.1

40.0

Male factor + tubal factor (%)

14.3

16.0

Ovulation failure (%)

0.0

4.0

Endometriosis (%)

4.8

4.0

14.3

16.0

Idiopathic (%)

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Table 2 Reasons for cancelled IVF-ET cycles

Reasons for cycles cancellation
Failed oocytes retrieval

RSDP group
4

RLDP group
2

Unfertilization

Unavailable embryos

Thin endometrium

Prevention of OHSS by whole embryo freezing

Embryo transfer failure

OHSS: ovarian hyperstimulation syndrome

average age in RSDP group was older than that in RLDP group by about 4 years with
significant difference (P<0.05), and the basal FSH level was higher in RSDP group than in
RLDP group (P<0.05).
As shown in Table 3, the Gn dosage had no significant difference between the two
groups (P>0.05), but Gn duration in RSDP group was significantly shorter than that in RLDP
group. The E2, LH and P levels on hCG injection day were lower in RSDP group than in
RLDP group with statistical significance. The number of retrieved oocytes and mature
oocytes in the RSDP group was significantly lower (P<0.05), as compared with those in
RLDP group, but there was no statistical difference for fertilization rate (P>0.05). Moreover,
both the good-quality embryos rate and implantation rate were significantly higher in RSDP
group as compared with those in RLDP group (P<0.05), regardless of the significantly
lower valid embryos in the former (P<0.05). More clinical pregnancies with fetal heart
activity were achieved in RSDP group than that in RLDP group (P<0.05).

Discussion
Routine down regulation regime had been commonly used for most infertile women.
Pituitary down regulation by GnRHa injection at mid-luteal phase could effectively inhibit
endogenous and premature LH surge and prevent the premature luteinization of oocytes.
But the pregnancy rate resulted from RLDP and other various protocols were not satisfied
for patients with repeated implantation failure. Women who experienced repeated IVF failure
with implantation barrier presented a challenging and frustrating problem. It was well
accepted that successful embryo implantation depended on the two key factors of embryo
quality and endometrium receptivity. So improvement on embryo quality and endometrium
receptivity would lead to enhancement in implantation rate and pregnancy rate. Various
treatment protocols have been proposed targeting at this particular cohort of women now.
These protocols can be reviewed as followed:
1) Hysteroscopy performed to find and treat RIF anatomic causes.
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Table 3 IVF-ET outcomes between the two groups

Item
Cycles cancellation (%)
ET cycles (n)

RSDP group

RLDP group

19.2 (10/52)

16.7 (10/60)

42

50

IVF cycles (%)

71.4 (30/42)

60.0 (30/50)

ICSI cycles (%)

28.6 (12/42)

40.0 (20/50)

Gn dosage ( IU)
Gn duration (d)

2 297 599

2 172 758

P
>0.050
>0.050

>0.050

10.1 1.7

11.4 1.8

0.001

E2 on hCG injection day (pg/ml)

1 428 792

2 954 1 380

<0.001

LH on hCG injection day (IU/ml)

0.97 0.51

1.40 0.69

0.002

P on hCG injection day (ng/ml)

0.77 0.37

1.04 0.62

0.015

Endometrial thickness on hCG injection day (mm)

11.7 3.0

11.9 3.0

>0.050

Number of retrieved oocytes (n)

6.05 3.22

10.96 5.78

<0.001

Number of mature oocytes (n)

4.62 2.70

9.24 5.39

<0.001

IVF cycles

76.07 21.74

75.71 21.38

>0.050

ICSI cycles

97.22 6.50

95.14 10.16

>0.050
0.006

Fertilization rate (%)

Number of valid embryos (n)

3.0 2.0

4.6 3.4

Good-quality embryo rate (%)

82.22 28.48

61.37 34.21

0.002

Number of embryos transferred (n)

2.0 0.7

2.0 0.5

>0.050

Implantation rate (%)

39.0 (32/82)

22.4 (22/98)

0.001

Clinical pregnancy rate (%)

57.1 (24/42)

32.0 (16/50)

0.015

0.0 (0/52)

10.0 (6/60)

0.035

Moderate to severe OHSS occurance (%)

2) The application of oral or vaginal estrogen pills, aspirin, and other medications that may
increase blood flow to the endometrium .
3) Mechanical endometrial stimulation performed in the cycle preceding the actual
treatment cycle to induce an inflammatory response that may facilitate the preparation of the
endometrium for implantation[3-5].
4) Locally or systemically administered human leukemia inhibitory factor or granulocyte
colony-stimulating factor to improve IVF outcome in patients with unexplained RIF[6-8].
5) Treatment with low molecular weight heparin (LMWH) or a mini-dose of aspirin
and/or corticosteroids for thrombophilia and connective tissue disease[9,10].
6) High-dose IV immunoglobulin offered before ET for immunologic factor[11].
7) Intracytoplasmic morphologically selected sperm injection (IMSI) for those with a
high percentage of abnormal sperm cells[12,13].
8) Measures to ameliorate the embryonic factors such as zygote intrafallopian transfer[14],
blastocyst transfer, sequential embryo transfer[15], and embryo coculture[16].
9) Preimplantation genetic diagnosis (PGD) for genetic causes.
Since protocol of COH played pivotal role in the improvement of embryo quality and
implantation, and pregnancy rate of the routine protocols was not satisfying in patients with
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RIF, we used RSDP on these patients and demonstrated that pregnancy rate of RSDP was
much higher than that of RLDP at the same period of time. Although the number of retrieved
oocytes and valid embryos was significantly lower in RSDP than in RLDP, the good-quality
embryos rate and implantation rate both were significantly higher in the former than those in
the latter. The levels of E2, LH and P on hCG injection day in RSDP was lower than those
in RLDP especially with statistical significance. We speculated that less follicles in the cohort
were recruited by RSDP resulting that lower levels of estradial were produced and less
oocytes and embryos were achieved. These results dont agree with those of Zhang Hui-juan[17]
who compared between the two protocols in patients with poor response, mainly because
their RSDP and the according subjects were completely different from ours. In our results,
RSDP attained to the satisfying pregnancy rate of 57.1%, as was much higher than that of
RLDP with the 32% pregnancy rate although the patients average age was elder by about
4 years in RSDP group compared with RLDP group.
We presumed the reasons for the improvement of pregnancy rate in RIF patients were
as followed. Firstly, the internal milieu was changed to enhance the endometrium receptivity.
RSDP management was similar to that of hypogonadotrophism, and it helped to inhibit irregular
hyperplasia and inflammation of endometrium to be in favour of synchronization between
follicle development and endometrium. Moreover, the abnormal internal environment caused
by endometriosis could also be significantly inhibited, since the application of GnRHa in
endometriosis patients could not only relieve the symptoms, but also inhibit the endometriosis
lesion and improve the pelvic and ovary microenvironment, leading to amelioration of
reproductive capacity. Secondly, RSDP could significantly improve the quality of embryos.
Higher excellent embryo rate was obtained despite the low oocytes and embryos achievement
in RSDP. It was obvious that injection of GnRHa could inhibit the hypothalamic-pituitaryovarian (H-P-O) axis and the secretion of LH leading to the decrease of androgen, E2 and P
production and secretion. The decrease of androgen level might reduce the adverse effects
on the follicle development and avoid the excessive recruitment of follicles, especially in
PCOS[18]. In addition, the relatively low P level on hCG injection day might ameliorate the
receptivity of the endometria and improve the pregnancy rates. In the process of COH, the
effect of LH level at follicular phase on the outcome of ART was not confirmed. It is generally
accepted that excessive LH at follicular phase or premature LH surge might cause abnormal
development of follicles and inhibition of the secretion of meiosis inhibition factor (MIF) by
oocytes, and then cause the failure of fertilization because of over-maturation of the oocytes.
Besides, the high level of LH could induce the elevation of estrogen and the P receptor
appeared prematurely on the endometrium. The premature transformation of the endometria
to secretory phase had adverse effect on embryo implantation[19]. So well-controlled LH and
P levels on hCG injection day would contribute to the improvement of pregnancy rates. We
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supposed that RSDP could provide complete pituitary down-regulation and concordant
endocrinal environment for follicular development, as leading to the high excellent embryo
rate in these RIF patients.
In RSDP, the gonadotropin dosage was not increased but the duration of medication
was decreased significantly compared with RLDP, which proved that RSDP was economical
of time and money.
It is necessary to clarify the ideal cycle number of GnRHa pretreatment and how
RSDP improved the outcome of IVF-ET in infertile patients. Super-long down-regulation
protocol had been routinely applied in presently, and multiple investigations supported that the
application of GnRHa for 3-6 cycles before IVF was necessary. Ma et al.[20] analyzed the
IVF outcome of endometriosis patients who underwent 2-3 cycles of 3.75 mg GnRHa before
IVF and found that the extended pretreatment of GnRHa would increase Gn dosage and
extend duration of COH although pregnancy rate was increased. It was discord with our
results. In our study, twice injection of small dose of GnRHa before the COH obtained the
remarkable clinical pregnancy rate of 57.1%. The reasons might be that we only use small
dosage of GnRHa, the over-depression of the H-P-O axis had been evaded, and the poor
response to COH had been also prevented.
In a word, the application of RSDP in patients of RIF would not only increase the
pregnancy rates, but also decrease the cost. We might consider this regimen as an effective
and economical assisted conception protocol. But the limited number in our study might
cause bias and a further large scale investigation is necessary.

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(Received on May 15, 2014)

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