- Email: [email protected]
The effect of endometrial scratch on natural-cycle cryopreserved embryo transfer outcomes: a randomized controlled study
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The effect of endometrial scratch on natural-cycle cryopreserved embryo transfer outcomes: a randomized controlled study
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Article
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Jennifer Sze Man Mak, Cathy Hoi Sze Chung, Jacqueline Pui Wah Chung *, Grace Wing Shan Kong, Sotirios H Saravelos, Lai Ping Cheung, Tin-Chiu Li Assisted Reproduction Technology Unit, Prince of Wales Hospital, The Chinese University of Hong Kong, Hong Kong
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Dr Jennifer Sze Man Mak is a subspecialist trainee in Reproductive Medicine accredited by the Royal College of Obstetricians and Gynaecologists. She is the Assistant Professor at the Department of Obstetrics & Gynaecology, The Chinese University of Hong Kong and is currently undergoing a 1-year overseas training period in the UK.
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KEY MESSAGE This prospective, double-blind, randomized controlled study on the evaluation of the implantation and pregnancy rate after endometrial scratch prior to natural-cycle cryopreserved embryo transfer found no beneficial effect in an unselected group of women. Further studies on its effect in women with recurrent implantation failure after IVF are warranted.
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A B S T R A C T
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The benefit of endometrial scratch (ES) prior to embryo transfer is controversial. Systemic analysis has confirmed its potential benefit, especially in women with repeated IVF failures, yet most studies have focused on fresh embryo transfer, and its effect on vitrified-warmed embryo transfer (FET) cycles is yet to be explored. We hereby present our prospective, double-blind, randomized controlled study on the evaluation of the implantation and pregnancy rate after ES prior to natural-cycle FET. A total of 299 patients underwent natural-cycle FET and were randomized to receive ES (n = 115) or endocervical manipulation as control (n = 114) prior to FET cycle, and a total of 196 patients had embryo transfer (93 patients in each group). Our study showed no significant difference in the implantation and pregnancy rate, as well as the clinical and ongoing pregnancy or live birth rates between the two groups. It appears that ES does not have any beneficial effect on an unselected group of women undergoing FET in natural cycles. Further studies on its effect in women with recurrent implantation failure after IVF are warranted. © 2017 Published by Elsevier Ltd on behalf of Reproductive Healthcare Ltd.
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* Corresponding author. E-mail address: [email protected] (JPW Chung). http://dx.doi.org/10.1016/j.rbmo.2017.04.004 1472-6483/© 2017 Published by Elsevier Ltd on behalf of Reproductive Healthcare Ltd. Please cite this article in press as: Jennifer Sze Man Mak, et al., The effect of endometrial scratch on natural-cycle cryopreserved embryo transfer outcomes: a randomized controlled study, Reproductive BioMedicine Online (2017), doi: 10.1016/j.rbmo.2017.04.004
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Introduction
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Despite medical advances in assisted reproductive technologies in the last decade, including selecting the best embryo to transfer at blastocyst stage or reducing chromosomally abnormal embryos by preimplantation genetic screening, the success rate of IVF remains modest, with clinical pregnancy rate per embryo transfer at 33.8% (European IVF-Monitoring Consortium (EIM) for the European Society of Human Q3 Reproduction and Embryology (ESHRE) et al., 2016). Implantation is the rate-limiting step for the success of IVF (Paulson et al., 1990). Successful implantation requires a receptive endometrium, a functional embryo at the blastocyst developmental stage and a synchronized dialogue between maternal and embryonic tissues (Simon et al., 2000). Generally, the probability of an embryo successfully implanting is approximately 30%, while its failures can be multifactorial. Recurrent implantation failures (RIF) may occur in 5–10% of women undergoing IVF cycles, and a significant proportion of this is related to endometrial receptivity. Endometrial scratch (ES) is one of several strategies proposed to improve endometrial receptivity. The use of ES to improve implantation rates in women undergoing IVF was first described in 2003 (Barash et al., 2003), which showed that ES doubles the rates of implantation, clinical pregnancy and live birth in women with RIF. Since then, the procedure has been further studied in women with previous IVF failures (Almog et al., 2010; Baum et al., 2012; El-Toukhy et al., 2012; Gibreel et al., 2015; Gnainsky et al., 2010; Karimzadeh et al., 2009; Narvekar et al., 2010; Potdar et al., 2012; Raziel et al., 2007; Singh et al., 2015). To date, the majority of studies have found beneficial effect of ES in women undergoing embryo transfer (Barash et al., 2003; Guven et al., 2014; Inal et al., 2012; Karimzadeh et al., 2009; Narvekar et al., 2010; Nastri et al., 2013; Raziel Q4 et al., 2007; Shohayeb and El-Khayat, 2012; Singh et al., 2015), whereas a few others could not confirm the benefit (Baum et al., 2012; Yeung et al., 2014). Three systematic reviews and a meta-analysis all concluded that there appeared to be a general beneficial effect of ES in IVF (El-Toukhy et al., 2012; Potdar et al., 2012). Among the 14 trials included in a recent updated Cochrane Review, 13 randomized controlled trials (RCT) studied endometrial injury during the menstrual cycle before embryo transfer, in which seven studies included women with previous IVF failures, five included women regardless of the number of previous IVF cycles and one included only women undergoing their first IVF cycle; the study found a favourable effect of ES with an increased clinical pregnancy rate based on pooled results from 13 RCT including 1972 women [relative risk (RR) 1.34, 95% confidence interval (CI) 1.21–1.61, P = 0.002) and an increase in live birth or ongoing pregnancy rate from nine RCT including 1496 women (RR 1.42, 95% CI 1.08–1.85, P = 0.01) (Nastri et al., 2015). Since the Cochrane Review was published, two additional RCT have been reported (Mahran et al., 2016; Singh et al., 2015). Singh et al. (2015) examined women with previous failed IVF-embryo transfer and found a significant increase in implantation rate after ES in patients while Mahran et al. (2016) studied patients undergoing their first IVF cycle and observed significant improvement in both implantation and live birth rate. The findings of these two studies were consistent with the conclusion reached in the Cochrane Review. However, it is noteworthy that all the studies analysed in the Cochrane Review and the two recent, additional RCT were all related to fresh embryo transfer cycles.
There are several important confounding variables affecting the outcomes of ES, including the numbers of previous IVF failures and the type of embryo transfer cycles, i.e. fresh embryo transfer in stimulated cycle, FET in natural or artificial cycle using hormone replacement therapy (HRT). Whilst studies have been performed in fresh embryo transfer cycles (Barash et al., 2003; Baum et al., 2012; Gibreel et al., 2015; Guven et al., 2014; Inal et al., 2012; Karimzadeh et al., 2009; Mahran et al., 2016; Narvekar et al., 2010; Nastri et al., 2013; Raziel et al., 2007; Shohayeb and El-Khayat, 2012; Singh et al., 2015; Yeung et al., 2014) and FET in HRT cycles (Aflatoonian et al., 2016; Dunne and Taylor, 2014), the possible impact of ES in natural-cycle FET has not been previously examined. Consequently, we conducted a prospective, double-blind, randomized controlled trial to evaluate the impact of local endometrial injury in the mid-luteal phase of the cycle immediately preceding natural-cycle FET.
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Materials and methods
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Study design
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This was a prospective, double-blind, randomized controlled study conducted in the Assisted Reproductive Unit of the Prince of Wales Hospital, Department of Obstetrics and Gynaecology of The Chinese University of Hong Kong, during the period of March 2013 to April 2016.
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Study population All patients scheduled for FET cycles using non-donor oocytes were assessed for eligibility. Women who had normal ovulation and were deemed suitable for natural-cycle FET were recruited to the trial. The exclusion criteria included those who had any uterine anomaly or pathology such as endometrial polyps, endometriomas larger than 4 cm or hydrosalpinx.
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Randomization After informed consent, patients were randomly allocated to either study group who would receive ES or a control group in which endocervical manipulation would be performed using computergenerated random numbers concealed in opaque envelopes. A research nurse coordinated the randomization process. The doctors who performed the ETs, the embryologists and the patients involved in the study were blinded to the treatment allocation.
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Intervention ES (study group) or endocervical manipulation (control group) were performed at the mid-luteal phase of the preceding menstrual cycle before FET, which was scheduled to take place 7 ± 1 days after the surge of LH, according to the LH surge identified by daily urine LH monitoring from day 8 of the cycle. Serum progesterone level was checked at the same time to confirm spontaneous ovulation.
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Endometrial biopsy Endometrial samples were obtained using a biopsy catheter (Pipette; MedGyn, USA) by inserting the pipette through the cervical os and
Please cite this article in press as: Jennifer Sze Man Mak, et al., The effect of endometrial scratch on natural-cycle cryopreserved embryo transfer outcomes: a randomized controlled study, Reproductive BioMedicine Online (2017), doi: 10.1016/j.rbmo.2017.04.004
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advancing gently until resistance was felt. The inner piton of the device was then withdrawn to create suction to obtain the endometrial sample by moving the pipette up and down approximately 2–3 cm within the uterine cavity. The procedure was repeated at least four times with the device being rotated 360° to ensure adequate coverage of the area.
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Endocervical manipulation
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A sterile cotton wool stick was inserted about 2 cm into the cervical os; the stick was moved up and down inside the cervical canal with 360° rotation to mimic the procedure of endometrial sampling.
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Sample size According to our own hospital’s audit, the mean pregnancy rate after FET was around 30%. Assuming that the pregnancy rate of FET following ES was 50%, and accepting an alpha value of 0.05 and 80% power the number of subjects in each arm of the study required would be 93 cases. Assuming that 10% of the patients would be excluded due to incomplete data or dropouts, the total required sample size was 206.
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Ethics approval
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Embryo cryopreservation
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Embryos were cryopreserved using the Vitrification Freeze Kit (Irvine Q5 Q6 Scientific 90133, USA) (Irvine Scientific, 2016a, http://www.irvinesci .com/simplified-embryo-vitrification-protocols) and warmed for subsequent embryo transfer using the Vitrification Thaw Kit (Irvine Q7 Scientific 90137, USA) (Irvine Scientific, 2016b, http://www.irvinesci .com/simplified-embryo-and-oocyte-warming-protocols) as per standard protocols.
The study was approved by the Joint Chinese University of Hong Kong – New Territories East Cluster Clinical Research Ethics Committee on 7 February 2012 (CREC Ref. No.: 2011.531-T) and was registered with the Chinese Clinical Trial Registry (Registration No.: ChiCTRTRC-12002389). Informed consents were obtained from all patients who participated in the study, which was conducted in accordance with the Declaration of Helsinki and Good Clinical Practice.
Statistical analysis Natural-cycle vitrified-warmed ET (FET)
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In the FET cycle, all patients were monitored for evidence of ovulation. In patients who had normal ovulation, serial hormonal assays (oestradiol and LH) were performed to determine the time of ovulation and the day of embryo transfer, depending on the stage of the embryo to be transferred. The couple would be advised to refrain from having unprotected intercourse during the FET cycle. In our unit during the period of study, the policy was to transfer one or two embryos. In exceptional circumstances, three embryos would be transferred, but this only occurred in fewer than 1% of cases. Embryos were assessed based on morphological criteria (number, size and shape of blastomeres, degree of fragmentation, appearance of cytoplasm) and were categorized into five grades (grade 1 [worse] to grade 5 [best]). Embryos of ≥4 cells on Day 2 and ≥7 cells on Day 3 with <10% fragmentation were regarded as good-quality embryos (grades 4 and 5). The better-quality embryos would be chosen for transfer first and if available, blastocysts would be transferred before the cleavage stage embryos. Luteal phase support was not given in all cases. A blood sample was taken 9 days after transfer of a blastocyst or 11 days after transfer of a cleavage stage embryo for human chorionic gonadotropin (HCG) measurement to verify if pregnancy had occurred. In women with a positive pregnancy test, a transvaginal ultrasound scan was performed 2 weeks later to determine the location of pregnancy, the number of gestational sacs and fetal viability.
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Outcome measures
All analyses were performed using the Statistical Package of Social Sciences for Mac Version 22 (IBM Corp., USA). Continuous data were assessed for normality of distribution using the Shapiro–Wilk test, and expressed as mean (±SD) or median (interquartile range) accordingly; comparisons were made using the Student’s t-test and the Mann–Whitney U-test, respectively. Categorical data were expressed as number (percentage); comparisons were made using the chi-squared test or Fisher’s exact test and presented with rate ratios and 95% CIs. A two-sided value of P < 0.05 was considered as significant for all statistical tests performed.
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Results Participant flow The CONSORT diagram in Figure 1 demonstrates the participant flow. A total of 845 women were assessed for eligibility, of which 229 were recruited and ultimately 186 completed the study.
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Baseline and cycle characteristics The baseline and cycle characteristics of the ES and control groups are presented in Table 1. There were no statistically significant differences between the two groups for all the variables presented.
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The primary outcome measure was pregnancy rate, which was defined as a positive urine pregnancy test per embryo transfer. Secondary outcome measures were implantation rate, which was defined as the number of gestational sacs seen on ultrasound examination per embryo transferred; clinical pregnancy rate, which referred to the number of pregnancies with confirmed intrauterine gestational sac; ongoing pregnancy rate, which referred to the presence of at least one fetus with heart pulsation proceeding beyond 32 weeks of gestation; and live birth rate which referred to the number of deliveries with at least one live-born infant.
Outcome analysis The outcome analysis of the study is presented in Table 2. There was no significant difference in the pregnancy rate (ES group 48.4% versus control group 43.0%; RR 1.125, 95% CI 0.822–1.540) and the implantation rate (ES group 36.5% versus control group 32.8%; RR 1.143, 95% CI 0.802–1.627) between the two groups. In addition, there were no significant differences in the secondary outcome measures including clinical pregnancy and ongoing pregnancy or live birth rates between the two groups.
Please cite this article in press as: Jennifer Sze Man Mak, et al., The effect of endometrial scratch on natural-cycle cryopreserved embryo transfer outcomes: a randomized controlled study, Reproductive BioMedicine Online (2017), doi: 10.1016/j.rbmo.2017.04.004
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Figure 1 – CONSORT 2010 flow diagram.
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The findings were similar when performing intention-to-treat analysis with no statistically significant difference in the pregnancy rate (ES group 39.1% versus control group 35.1%; RR 1.115, 95% CI 0.795– 1.564) and the implantation rate (ES group 36.5% versus control group 32%; RR 1.143, 95% CI 0.802–1.627) between the two groups.
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Subgroup analysis and logistic regression
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Subgroup analysis was performed by stratifying women into those with one or more, two or more, or three or more previous ETs and two or more failed IVF attempts. No statistically significant differences were detected between ES and control groups in any of the outcome measures for any of the stratified groups (Figures 2 and 3).
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Discussion
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To the best of our knowledge this is the first RCT to evaluate the impact of ES on clinical outcomes in women undergoing natural-cycle FET.
We found no statistically significant differences between the two groups in the various clinical outcomes. Endometrial injury is defined as intentional damage to the endometrium performed to improve endometrial receptivity and therefore the implantation and pregnancy rates. We are aware that there are many methods to induce endometrial injury and there is no universal standard of practice. We know some investigators use a more invasive approach such as hysteroscopy-induced endometrial injury (Huang et al., 2011; Shokeir et al., 2016). However, most investigators use a more gentle approach using ES by pipelle (Nastri et al., 2015). We used this approach in this study, and our observation based on this soft ES showed that it does not have any beneficial effect on an unselected group of women undergoing FET in natural cycles. It is possible that the degree of injury may have an impact on pregnancy outcomes. However, there is no consensus on the optimal method of endometrial injury so as to exert maximal effect. Further adequately powered studies would be needed to clarify this. Although the recently updated Cochrane Review concluded a favourable effect of ES with an increased clinical pregnancy rate and live birth or ongoing pregnancy rate with moderate quality evidence
Please cite this article in press as: Jennifer Sze Man Mak, et al., The effect of endometrial scratch on natural-cycle cryopreserved embryo transfer outcomes: a randomized controlled study, Reproductive BioMedicine Online (2017), doi: 10.1016/j.rbmo.2017.04.004
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Table 1 – Comparison of baseline and cycle characteristics between the two randomized groups. Characteristics
ES group (n = 93)
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Baseline characteristics
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65 (69.9)
62 (66.7)
Tuboperitoneal, n (%)
45 (48.4)
46 (49.5)
Endometriosis, n (%)
16 (17.2)
21 (22.6)
Male, n (%)
42 (45.2)
44 (47.3)
5 (5.4)
3 (3.2)
infertilityc,d,#
1–2, n (%)
85 (91.4)
88 (94.6)
3–4, n (%)
8 (8.6)
5 (5.4)
1–2, n (%)
48 (51.6)
58 (62.4)
≥3, n (%)
24 (25.8)
20 (21.5)
1–2, n (%)
62 (66.7)
66 (70.1)
3–4, n (%)
5 (5.4)
2 (2.2)
1–2, n (%)
29 (31.2)
29 (31.2)
≥3, n (%)
8 (8.6)
9 (9.7)
Agonist
54 (58.1%)
61 (65.6%)
Antagonist
39 (41.9%)
32 (34.4%)
Baseline FSH (IU/l)b,*
7.3 (6.4–8.1)
7.1 (6.1–8.3)
Total gonadotrophin used
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(IU)b,
Oestradiol on trigger day (pmol/l)b,*
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Total oocytes retrievedb,*
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Insemination
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Intracytoplasmic sperm injection, n (%)
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Fertilization rate (%)a,‡
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Viable embryos for cryopreservationb,*
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Endometrial thickness (mm)a,‡
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Survival rate of warmed embryos (%)e
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*
10.92 ± 1.43
10.91 ± 1.56
3525 (2475–4500)
3300 (2250–4312)
7051 (3987–13057)
7824 (3750–13818)
11 (9–12)
10 (8–12)
44 (47.3)
43 (46.2)
49 (52.7)
50 (53.8)
methodb,#
Conventional, n (%)
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74.88 ± 18.01
73.16 ± 17.54
4 (3–6)
4 (3–6)
11.48 ± 2.30
11.13 ± 2.35
118/125 (94.4)
123/135 (91.1)
Number of embryos transferredc,#
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1, n (%)
71 (76.3)
64 (68.8)
2, n (%)
22 (23.7)
29 (31.2)
Day 2/3, n (%)
37 (39.8)
44 (47.3)
Day 5/6, n (%)
56 (60.2)
49 (52.7)
50 (43.5)
63 (51.6)
Stage of embryo transferredc,#
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Good-quality embryos transferreda, n (%)
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There were no statistically significant differences between the two groups. BMI = body mass index; ES = endometrial scratch; ET = embryo transfer. a
Data are mean ± SD; comparisons performed using Student’s t-test.
b
Data are median (25th to 75th centiles); comparisons performed using Mann–
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‡
Whitney U-test.*
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Data are number (%); comparisons performed using chi-squared test# or Fisher’s exact test.†
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Some patients presented with more than one cause of infertility.
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Number of embryos survived after warming/number of embryos warmed.
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Secondary, n (%)
Duration of stimulation (days)a,‡
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31 (33.3)
Treatment protocolc,#
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28 (30.1)
Cycle characteristics
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Primary, n (%)
Number of previous frozen ETc,#
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4.73 ± 3.26
Number of previous fresh ETc,†
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21.90 ± 2.81
4.74 ± 3.62
Number of previous ETc,#
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21.36 ± 2.81
)
Number of previous IVF attemptsc,#
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BMI
Unexplained, n (%)
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36.78 ± 3.45
Cause of
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36.90 ± 3.32
(kg/m2 a,‡
Type of infertilityc,#
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Age
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Infertility duration (years)a,‡
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Non-ES group (n = 93)
5
(Nastri et al., 2015), many confounding variables have been recognized, including the number of previous IVF failures, the number and quality of embryos transferred and the timing of ES. In addition, it is likely that another confounding variable is the type of embryo transfer cycle. Whilst the majority of earlier studies which demonstrated a beneficial effect of ES were based on fresh embryo transfer cycles (Barash et al., 2003; Guven et al., 2014; Inal et al., 2012; Karimzadeh et al., 2009; Mahran et al., 2016; Narvekar et al., 2010; Nastri et al., 2013; Raziel et al., 2007; Shohayeb and El-Khayat, 2012; Singh et al., 2015), there have been three studies examining the impact of ES in FET cycles (Aflatoonian et al., 2016; Dunne and Taylor, 2014; Kanazawa et al., 2017), each of which appears to have different results. In the first study by Dunne and Taylor (2014), which was a prospective cohort study examining 80 women with at least one failed IVF-ET, ES was performed between day 21 and 23 of the menstrual cycle preceding HRT-FET, and no statistically significant differences in implantation or clinical pregnancy rates were found between those with ES and those without. The second study was a randomized study by Aflatoonian et al. (2016) with 45 women in the ES group and 48 women in the Q8 control group; ES was performed between day 21 and 23 of the menstrual cycle preceding HRT-FET. However, the implantation and clinical pregnancy rates appeared to be lower in the ES group, but the difference was not statistically significant. The implantation rate was 11.8% versus 20.5%, clinical pregnancy rate was 22.2% versus 33.3% and ongoing pregnancy rate was 22.2% versus 31.2% in the ES and control groups, respectively. In a third study by Kanazawa et al. (2017), which was a retrospective cohort study examining a total of 173 women, in which patients mostly underwent HRT-FET (150 women on HRTFET and 23 on natural-cycle FET) and were divided into three groups: Group A (n = 38) underwent soft curettage to the endometrium twice prior to the FET cycle; Group B (n = 45) underwent hysteroscopy prior to the FET cycle, with no significant factors such as endometrial polyps; and Group C (n = 90) was the control group. The study found a positive effect of ES in FET cycles, reporting a statistically significant higher clinical pregnancy rate per transfer between Group A and Group C (42.1% versus 22.2%, adjusted odds ratios 2.49, 95% CI 1.01–6.17, P = 0.048), while Group B with only hysteroscopy had a higher pregnancy rate than Group C but showed no statistical significance (35.6% versus 22.2%, adjusted odds ratios 2.14, 95% CI 0.89–5.15). Considering the three studies together, it is impossible to draw a firm conclusion regarding whether or not ES in HRT-FET cycles is of benefit, especially because only one of the three studies was a randomized trial and one of them consisted of predominantly HRT cycles with only a small number of natural cycles, which may be another confounding variable. As far as we are aware, our study was the first conducted on the impact of ES on FET in natural cycles. We found no significant differences between the two groups in the various pregnancy outcomes. Whilst current data suggests that the implantation and clinical pregnancy rates are similar between FET in HRT cycles and natural cycles (Ghobara and Vandekerckhove, 2008; Groenewoud et al., 2016), it is unclear whether the impact of ES in these two types of cycle is the same. It is not scientifically sound to extrapolate data from one type of FET cycle to another. In this respect, a particular strength of our study was that it included only one type of FET cycle, namely FET in natural cycles, unlike the study by Kanazawa et al. (2017) which included a mixture of HRT (majority) and natural cycles. On the other hand, one possible criticism of our study, similar to that of Yeung et al. (2014), was the inclusion of patients with a variable number of previous IVF or embryo transfer attempts. It seems
Please cite this article in press as: Jennifer Sze Man Mak, et al., The effect of endometrial scratch on natural-cycle cryopreserved embryo transfer outcomes: a randomized controlled study, Reproductive BioMedicine Online (2017), doi: 10.1016/j.rbmo.2017.04.004
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Table 2 – Comparison of pregnancy outcomes per transfer between the two randomized groups (per protocol analysis). Parameter
ES group (n = 93)
Non-ES group (n = 93)
Relative risk 95% CI
Positive HCG ratea, n (%) Biochemical pregnancy ratea, n (%) Implantation ratea, n (%) Clinical pregnancy ratea, n (%) Miscarriage ratea, n (%) Multiple pregnancy ratea, n (%) Ongoing pregnancy/live birth ratea, n (%)
45/93 6/93 42/115 39/93 7/39 4/39 32/93
40/93 5/93 39/122 35/93 6/35 4/35 29/93
1.125 1.200 1.143 1.114 0.946 0.897 1.158
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(48.4) (6.5) (36.5) (41.9) (17.9) (10.3) (34.4)
(43.0) (5.4) (32.0) (37.6) (17.1) (11.4) (31.2)
(0.822–1.540) (0.379–3.795) (0.802–1.627) (0.782–1.588) (0.285–3.142) (0.242–3.321) (0.627–2.137)
ES = endometrial scratch; HCG = human chorionic gonadotrophin. a Data are number (%); comparisons performed using chi-squared test. There were no statistically significant differences between the two groups.
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that ES is more likely to have a beneficial effect in women with two or more ET failures compared with an unselected group of subjects undergoing IVF-ET (Nastri et al., 2015). Although it is possible to perform a subgroup analysis of subjects with regard to the number of previous failures, as in the case of our current study and that of Yeung et al. (2014), the subgroup analysis inevitably lacks sufficient power. In this respect, what we can conclude from this study is that ES does not appear to have any beneficial effect on an unselected group of women undergoing FET in natural cycles; but it is still uncertain whether or not ES has any benefit in women with recurrent implantation failure after IVF. Our power calculation was performed based on three previous meta-analyses in 2012, which reported at least a 50% increase in pregnancy rate after endometrial injury in the cycle prior to IVF (El-Toukhy et al., 2012; Nastri et al., 2012; Potdar et al., 2012). With the benefit of hindsight, the earlier estimate of an increase in pregnancy rate of FET from 30% to 50% following ES could well represent an overestimate. In order to remove bias as a double-blind RCT, we performed endocervical manipulation as control, on the assumption that cervical stimulation would not affect the outcome (if it did, it would be negligible because the procedure was confined to the cervical canal and should not disturb the endometrial environment). The exact mechanism by which ES may improve implantation remains unclear. Several possible mechanisms have been proposed (Dekel et al., 2010; Gnainsky et al., 2010; Kalma et al., 2009; Zhou et al., 2008). Whatever the underlying mechanism, it is highly plausible that the benefit, if any, of ES depends on the pre-existing condition of the endometrium. The current observation that ES is more likely to have a beneficial effect in women with recurrent implantation failure than an unselected group of subjects lends support to such a notion as women with RIF are considered to be more likely to exhibit abnormal endometrial features (Makrigiannakis et al., 2011). In this context, it is interesting to note that the implantation rate of fresh embryo transfer is generally considered to be inferior to vitrified-warmed embryo transfer because the endometrium in fresh embryo transfer cycles often exhibits dis-synchrony because of the supra-physiological levels of steroid hormones encountered in hyper-stimulated cycles (Bourgain and Devroey, 2003; Check et al., 1999; Devroey et al., 2004; Nikas et al., 1999; Ochsenkuhn et al., 2012; Richter et al., 2006; Roque, 2015; Roque et al., 2013, 2015; Shapiro et al., 2014), especially if the progesterone level on the day of HCG trigger is significantly elevated (Papanikolaou et al., 2012; Santos-Ribeiro et al., 2014; Venetis et al., 2013). It is tempting to speculate that ES is more likely to have beneficial effects in an abnormally developing endometrium (in stimulated cycles) than normally developing ones (in natural cycles).
Some investigators have speculated that ES may have adverse effects (Aflatoonian et al., 2016). Whilst we have found that ES did not have any beneficial effect, it did not seem to have any adverse impact either, with implantation and pregnancy rates very similar between those who did and did not have ES.
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Conclusion
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ES in the luteal phase of the cycle preceding the natural-cycle FET in an unselected group of women does not improve implantation and pregnancy rates.
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Acknowledgements
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The authors wish to thank research nurses Ms Eris Tse and Ms Cosy Cheung for helping with patient recruitment, randomization and data entry; Ms Pauline Chan for her valuable contribution to the data collection; Ms Mei-Chun Cheung, Ms Stella Tang and Ms Janet Lau in the ART unit for their help during the study. Finally, we are grateful to all the patients attending our unit for their willingness to participate in this study.
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A R T I C L E
I N F O
Article history: Received 2 December 2016 Received in revised form 4 April 2017 Accepted 5 April 2017
Declaration: The authors report no financial or commercial conflicts of interest.
Keywords: Endometrial injury Endometrial scratch Implantation Natural cycle Pregnancy rate Vitrified-warmed embryo transfer
Please cite this article in press as: Jennifer Sze Man Mak, et al., The effect of endometrial scratch on natural-cycle cryopreserved embryo transfer outcomes: a randomized controlled study, Reproductive BioMedicine Online (2017), doi: 10.1016/j.rbmo.2017.04.004
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Figure 2 – Subgroup analysis: Comparison of pregnancy outcomes between the two randomized groups with one or more, two or more and three or more previous embryo transfers. (A) One or more previous embryo transfers. There were no statistically significant differences between the two groups. (B) Two or more previous embryo transfers. There were no statistically significant differences between the two groups. (C) Three or more previous embryo transfers. There were no statistically significant differences between the two groups.
Please cite this article in press as: Jennifer Sze Man Mak, et al., The effect of endometrial scratch on natural-cycle cryopreserved embryo transfer outcomes: a randomized controlled study, Reproductive BioMedicine Online (2017), doi: 10.1016/j.rbmo.2017.04.004
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Figure 3 – Subgroup analysis: comparison of pregnancy outcomes between the two randomized groups with two or more failed IVF
Q10 attempts. There were no statistically significant differences between the two groups.
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Kanazawa, E., Nakashima, A., Yonemoto, K., Otsuka, M., Yoshioka, N., Kuramoto, T., Mitao, H., Imaishi, H., Komai, K., Ushijima, K., 2017. Injury to the endometrium prior to the frozen-thawed embryo transfer cycle improves pregnancy rates in patients with repeated implantation failure. J. Obstet. Gynaecol. Res. 43, 128–134. Karimzadeh, M.A., Ayazi Rozbahani, M., Tabibnejad, N., 2009. Endometrial local injury improves the pregnancy rate among recurrent implantation failure patients undergoing in vitro fertilisation/intra cytoplasmic sperm injection: a randomised clinical trial. Aust. N. Z. J. Obstet. Gynaecol. 49, 677–680. Mahran, A., Ibrahim, M., Bahaa, H., 2016. The effect of endometrial injury on first cycle IVF/ICSI outcome: a randomized controlled trial. Int. J. Reprod. Biomed. (Yazd) 14, 193–198. Makrigiannakis, A., Petsas, G., Toth, B., Relakis, K., Jeschke, U., 2011. Recent advances in understanding immunology of reproductive failure. J. Reprod. Immunol. 90, 96–104. Narvekar, S.A., Gupta, N., Shetty, N., Kottur, A., Srinivas, M., Rao, K.A., 2010. Does local endometrial injury in the nontransfer cycle improve the IVF-ET outcome in the subsequent cycle in patients with previous unsuccessful IVF? A randomized controlled pilot study. J. Hum. Reprod. Sci. 3, 15–19. Nastri, C.O., Gibreel, A., Raine-Fenning, N., Maheshwari, A., Ferriani, R.A., Bhattacharya, S., Martins, W.P., 2012. Endometrial injury in women undergoing assisted reproductive techniques. Cochrane Database Syst. Rev. (7), CD009517. Nastri, C.O., Teixeira, D.M., Martins, W.P., 2013. Endometrial injury in the menstrual cycle prior to assisted reproduction techniques to improve reproductive outcomes. Gynecol. Endocrinol. 29, 401–402. Nastri, C.O., Lensen, S., Polanski, L., Raine-Fenning, N., Farquhar, C.M., Martins, W.P., 2015. Endometrial injury in women undergoing assisted reproductive techniques. Cochrane Database Syst. Rev. (3), CD009517. Nikas, G., Develioglu, O.H., Toner, J.P., Jones, H.W., Jr., 1999. Endometrial pinopodes indicate a shift in the window of receptivity in ivf cycles. Hum. Reprod. 14, 787–792. Ochsenkuhn, R., Arzberger, A., von Schonfeldt, V., Gallwas, J., Rogenhofer, N., Crispin, A., Thaler, C.J., Noss, U., 2012. Subtle progesterone rise on the day of human chorionic gonadotropin administration is associated with lower live birth rates in women undergoing assisted reproductive technology: a retrospective study with 2,555 fresh embryo transfers. Fertil. Steril. 98, 347–354. Papanikolaou, E.G., Pados, G., Grimbizis, G., Bili, E., Kyriazi, L., Polyzos, N.P., Humaidan, P., Tournaye, H., Tarlatzis, B., 2012. GnRH-agonist versus GnRH -antagonist IVF cycles: is the reproductive outcome affected by the incidence of progesterone elevation on the day of hCG triggering? A randomized prospective study. Hum. Reprod. 27, 1822–1828. Paulson, R.J., Sauer, M.V., Lobo, R.A., 1990. Factors affecting embryo implantation after human in vitro fertilization: a hypothesis. Am. J. Obstet. Gynecol. 163, 2020–2023. Potdar, N., Sauer, M.V., Lobo, R.A., 2012. Endometrial injury to overcome recurrent embryo implantation failure: a systematic review and meta-analysis. Reprod. Biomed. Online 25, 561–571.
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Raziel, A., Schachter, M., Strassburger, D., Bern, O., Ron-El, R., Friedler, S., 2007. Favorable influence of local injury to the endometrium in intracytoplasmic sperm injection patients with high-order implantation failure. Fertil. Steril. 87, 198–201. Richter, K.S., Shipley, S.K., McVearry, I., Tucker, M.J., Widra, E.A., 2006. Cryopreserved embryo transfers suggest that endometrial receptivity may contribute to reduced success rates of later developing embryos. Fertil. Steril. 86, 862–866. Roque, M., 2015. Freeze-all policy: is it time for that? J. Assist. Reprod. Genet. 32, 171–176. Roque, M., Lattes, K., Serra, S., Sola, I., Geber, S., Carreras, R., Checa, M.A., 2013. Fresh embryo transfer versus frozen embryo transfer in in vitro fertilization cycles: a systematic review and meta-analysis. Fertil. Steril. 99, 156–162. Roque, M., Valle, M., Guimaraes, F., Sampaio, M., Geber, S., 2015. Freeze-all policy: fresh vs. Frozen-thawed embryo transfer. Fertil. Steril. 103, 1190–1193. Santos-Ribeiro, S., Polyzos, N.P., Haentjens, P., Smitz, J., Camus, M., Tournaye, H., Blockeel, C., 2014. Live birth rates after IVF are reduced by both low and high progesterone levels on the day of human chorionic gonadotrophin administration. Hum. Reprod. 29, 1698–1705. Shapiro, B.S., Daneshmand, S.T., Garner, F.C., Aguirre, M., Hudson, C., 2014. Clinical rationale for cryopreservation of entire embryo cohorts in lieu of fresh transfer. Fertil. Steril. 102, 3–9. Shohayeb, A., El-Khayat, W., 2012. Does a single endometrial biopsy regimen (S-Ebr) improve ICSI outcome in patients with repeated implantation failure? A randomised controlled trial. Eur. J. Obstet. Gynecol. Reprod. Biol. 164, 176–179. Shokeir, T., Ebrahim, M., El-Mogy, H., 2016. Hysteroscopic-guided local endometrial injury does not improve natural cycle pregnancy rate in women with unexplained infertility: randomized controlled trial. J. Obstet. Gynaecol. Res. 42, 1553–1557. Simon, C., Martin, J.C., Pellicer, A., 2000. Paracrine regulators of implantation. Baillieres Best Pract. Res. Clin. Obstet. Gynaecol. 14, 815–826. Singh, N., Toshyan, V., Kumar, S., Vanamail, P., Madhu, M., 2015. Does endometrial injury enhances implantation in recurrent in-vitro fertilization failures? A prospective randomized control study from tertiary care center. J. Hum. Reprod. Sci. 8, 218– 223. Venetis, C.A., Kolibianakis, E.M., Bosdou, J.K., Tarlatzis, B.C., 2013. Progesterone elevation and probability of pregnancy after ivf: a systematic review and meta-analysis of over 60 000 cycles. Hum. Reprod. Update 19, 433–457. Yeung, T.W., Chai, J., Li, R.H., Lee, V.C., Ho, P.C., Ng, E.H., 2014. The effect of endometrial injury on ongoing pregnancy rate in unselected subfertile women undergoing in vitro fertilization: a randomized controlled trial. Hum. Reprod. 29, 2474–2481. Zhou, L., Li, R., Wang, R., Huang, H.X., Zhong, K., 2008. Local injury to the endometrium in controlled ovarian hyperstimulation cycles improves implantation rates. Fertil. Steril. 89, 1166–1176.
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The effect of endometrial scratch on natural-cycle cryopreserved embryo transfer outcomes: a randomized controlled study
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Jennifer Sze Man Mak, Cathy Hoi Sze Chung, Jacqueline Pui Wah Chung *, Grace Wing Shan Kong, Sotirios H Saravelos, Lai Ping Cheung, Tin-Chiu Li Assisted Reproduction Technology Unit, Prince of Wales Hospital, The Chinese University of Hong Kong, Hong Kong
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Dr Jennifer Sze Man Mak is a subspecialist trainee in Reproductive Medicine accredited by the Royal College of Obstetricians and Gynaecologists. She is the Assistant Professor at the Department of Obstetrics & Gynaecology, The Chinese University of Hong Kong and is currently undergoing a 1-year overseas training period in the UK.
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KEY MESSAGE This prospective, double-blind, randomized controlled study on the evaluation of the implantation and pregnancy rate after endometrial scratch prior to natural-cycle cryopreserved embryo transfer found no beneficial effect in an unselected group of women. Further studies on its effect in women with recurrent implantation failure after IVF are warranted.
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A B S T R A C T
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The benefit of endometrial scratch (ES) prior to embryo transfer is controversial. Systemic analysis has confirmed its potential benefit, especially in women with repeated IVF failures, yet most studies have focused on fresh embryo transfer, and its effect on vitrified-warmed embryo transfer (FET) cycles is yet to be explored. We hereby present our prospective, double-blind, randomized controlled study on the evaluation of the implantation and pregnancy rate after ES prior to natural-cycle FET. A total of 299 patients underwent natural-cycle FET and were randomized to receive ES (n = 115) or endocervical manipulation as control (n = 114) prior to FET cycle, and a total of 196 patients had embryo transfer (93 patients in each group). Our study showed no significant difference in the implantation and pregnancy rate, as well as the clinical and ongoing pregnancy or live birth rates between the two groups. It appears that ES does not have any beneficial effect on an unselected group of women undergoing FET in natural cycles. Further studies on its effect in women with recurrent implantation failure after IVF are warranted. © 2017 Published by Elsevier Ltd on behalf of Reproductive Healthcare Ltd.
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* Corresponding author. E-mail address: [email protected] (JPW Chung). http://dx.doi.org/10.1016/j.rbmo.2017.04.004 1472-6483/© 2017 Published by Elsevier Ltd on behalf of Reproductive Healthcare Ltd. Please cite this article in press as: Jennifer Sze Man Mak, et al., The effect of endometrial scratch on natural-cycle cryopreserved embryo transfer outcomes: a randomized controlled study, Reproductive BioMedicine Online (2017), doi: 10.1016/j.rbmo.2017.04.004
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Introduction
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Despite medical advances in assisted reproductive technologies in the last decade, including selecting the best embryo to transfer at blastocyst stage or reducing chromosomally abnormal embryos by preimplantation genetic screening, the success rate of IVF remains modest, with clinical pregnancy rate per embryo transfer at 33.8% (European IVF-Monitoring Consortium (EIM) for the European Society of Human Q3 Reproduction and Embryology (ESHRE) et al., 2016). Implantation is the rate-limiting step for the success of IVF (Paulson et al., 1990). Successful implantation requires a receptive endometrium, a functional embryo at the blastocyst developmental stage and a synchronized dialogue between maternal and embryonic tissues (Simon et al., 2000). Generally, the probability of an embryo successfully implanting is approximately 30%, while its failures can be multifactorial. Recurrent implantation failures (RIF) may occur in 5–10% of women undergoing IVF cycles, and a significant proportion of this is related to endometrial receptivity. Endometrial scratch (ES) is one of several strategies proposed to improve endometrial receptivity. The use of ES to improve implantation rates in women undergoing IVF was first described in 2003 (Barash et al., 2003), which showed that ES doubles the rates of implantation, clinical pregnancy and live birth in women with RIF. Since then, the procedure has been further studied in women with previous IVF failures (Almog et al., 2010; Baum et al., 2012; El-Toukhy et al., 2012; Gibreel et al., 2015; Gnainsky et al., 2010; Karimzadeh et al., 2009; Narvekar et al., 2010; Potdar et al., 2012; Raziel et al., 2007; Singh et al., 2015). To date, the majority of studies have found beneficial effect of ES in women undergoing embryo transfer (Barash et al., 2003; Guven et al., 2014; Inal et al., 2012; Karimzadeh et al., 2009; Narvekar et al., 2010; Nastri et al., 2013; Raziel Q4 et al., 2007; Shohayeb and El-Khayat, 2012; Singh et al., 2015), whereas a few others could not confirm the benefit (Baum et al., 2012; Yeung et al., 2014). Three systematic reviews and a meta-analysis all concluded that there appeared to be a general beneficial effect of ES in IVF (El-Toukhy et al., 2012; Potdar et al., 2012). Among the 14 trials included in a recent updated Cochrane Review, 13 randomized controlled trials (RCT) studied endometrial injury during the menstrual cycle before embryo transfer, in which seven studies included women with previous IVF failures, five included women regardless of the number of previous IVF cycles and one included only women undergoing their first IVF cycle; the study found a favourable effect of ES with an increased clinical pregnancy rate based on pooled results from 13 RCT including 1972 women [relative risk (RR) 1.34, 95% confidence interval (CI) 1.21–1.61, P = 0.002) and an increase in live birth or ongoing pregnancy rate from nine RCT including 1496 women (RR 1.42, 95% CI 1.08–1.85, P = 0.01) (Nastri et al., 2015). Since the Cochrane Review was published, two additional RCT have been reported (Mahran et al., 2016; Singh et al., 2015). Singh et al. (2015) examined women with previous failed IVF-embryo transfer and found a significant increase in implantation rate after ES in patients while Mahran et al. (2016) studied patients undergoing their first IVF cycle and observed significant improvement in both implantation and live birth rate. The findings of these two studies were consistent with the conclusion reached in the Cochrane Review. However, it is noteworthy that all the studies analysed in the Cochrane Review and the two recent, additional RCT were all related to fresh embryo transfer cycles.
There are several important confounding variables affecting the outcomes of ES, including the numbers of previous IVF failures and the type of embryo transfer cycles, i.e. fresh embryo transfer in stimulated cycle, FET in natural or artificial cycle using hormone replacement therapy (HRT). Whilst studies have been performed in fresh embryo transfer cycles (Barash et al., 2003; Baum et al., 2012; Gibreel et al., 2015; Guven et al., 2014; Inal et al., 2012; Karimzadeh et al., 2009; Mahran et al., 2016; Narvekar et al., 2010; Nastri et al., 2013; Raziel et al., 2007; Shohayeb and El-Khayat, 2012; Singh et al., 2015; Yeung et al., 2014) and FET in HRT cycles (Aflatoonian et al., 2016; Dunne and Taylor, 2014), the possible impact of ES in natural-cycle FET has not been previously examined. Consequently, we conducted a prospective, double-blind, randomized controlled trial to evaluate the impact of local endometrial injury in the mid-luteal phase of the cycle immediately preceding natural-cycle FET.
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Materials and methods
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Study design
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This was a prospective, double-blind, randomized controlled study conducted in the Assisted Reproductive Unit of the Prince of Wales Hospital, Department of Obstetrics and Gynaecology of The Chinese University of Hong Kong, during the period of March 2013 to April 2016.
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Study population All patients scheduled for FET cycles using non-donor oocytes were assessed for eligibility. Women who had normal ovulation and were deemed suitable for natural-cycle FET were recruited to the trial. The exclusion criteria included those who had any uterine anomaly or pathology such as endometrial polyps, endometriomas larger than 4 cm or hydrosalpinx.
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Randomization After informed consent, patients were randomly allocated to either study group who would receive ES or a control group in which endocervical manipulation would be performed using computergenerated random numbers concealed in opaque envelopes. A research nurse coordinated the randomization process. The doctors who performed the ETs, the embryologists and the patients involved in the study were blinded to the treatment allocation.
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Intervention ES (study group) or endocervical manipulation (control group) were performed at the mid-luteal phase of the preceding menstrual cycle before FET, which was scheduled to take place 7 ± 1 days after the surge of LH, according to the LH surge identified by daily urine LH monitoring from day 8 of the cycle. Serum progesterone level was checked at the same time to confirm spontaneous ovulation.
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Endometrial biopsy Endometrial samples were obtained using a biopsy catheter (Pipette; MedGyn, USA) by inserting the pipette through the cervical os and
Please cite this article in press as: Jennifer Sze Man Mak, et al., The effect of endometrial scratch on natural-cycle cryopreserved embryo transfer outcomes: a randomized controlled study, Reproductive BioMedicine Online (2017), doi: 10.1016/j.rbmo.2017.04.004
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advancing gently until resistance was felt. The inner piton of the device was then withdrawn to create suction to obtain the endometrial sample by moving the pipette up and down approximately 2–3 cm within the uterine cavity. The procedure was repeated at least four times with the device being rotated 360° to ensure adequate coverage of the area.
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Endocervical manipulation
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A sterile cotton wool stick was inserted about 2 cm into the cervical os; the stick was moved up and down inside the cervical canal with 360° rotation to mimic the procedure of endometrial sampling.
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Sample size According to our own hospital’s audit, the mean pregnancy rate after FET was around 30%. Assuming that the pregnancy rate of FET following ES was 50%, and accepting an alpha value of 0.05 and 80% power the number of subjects in each arm of the study required would be 93 cases. Assuming that 10% of the patients would be excluded due to incomplete data or dropouts, the total required sample size was 206.
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Ethics approval
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Embryo cryopreservation
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Embryos were cryopreserved using the Vitrification Freeze Kit (Irvine Q5 Q6 Scientific 90133, USA) (Irvine Scientific, 2016a, http://www.irvinesci .com/simplified-embryo-vitrification-protocols) and warmed for subsequent embryo transfer using the Vitrification Thaw Kit (Irvine Q7 Scientific 90137, USA) (Irvine Scientific, 2016b, http://www.irvinesci .com/simplified-embryo-and-oocyte-warming-protocols) as per standard protocols.
The study was approved by the Joint Chinese University of Hong Kong – New Territories East Cluster Clinical Research Ethics Committee on 7 February 2012 (CREC Ref. No.: 2011.531-T) and was registered with the Chinese Clinical Trial Registry (Registration No.: ChiCTRTRC-12002389). Informed consents were obtained from all patients who participated in the study, which was conducted in accordance with the Declaration of Helsinki and Good Clinical Practice.
Statistical analysis Natural-cycle vitrified-warmed ET (FET)
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In the FET cycle, all patients were monitored for evidence of ovulation. In patients who had normal ovulation, serial hormonal assays (oestradiol and LH) were performed to determine the time of ovulation and the day of embryo transfer, depending on the stage of the embryo to be transferred. The couple would be advised to refrain from having unprotected intercourse during the FET cycle. In our unit during the period of study, the policy was to transfer one or two embryos. In exceptional circumstances, three embryos would be transferred, but this only occurred in fewer than 1% of cases. Embryos were assessed based on morphological criteria (number, size and shape of blastomeres, degree of fragmentation, appearance of cytoplasm) and were categorized into five grades (grade 1 [worse] to grade 5 [best]). Embryos of ≥4 cells on Day 2 and ≥7 cells on Day 3 with <10% fragmentation were regarded as good-quality embryos (grades 4 and 5). The better-quality embryos would be chosen for transfer first and if available, blastocysts would be transferred before the cleavage stage embryos. Luteal phase support was not given in all cases. A blood sample was taken 9 days after transfer of a blastocyst or 11 days after transfer of a cleavage stage embryo for human chorionic gonadotropin (HCG) measurement to verify if pregnancy had occurred. In women with a positive pregnancy test, a transvaginal ultrasound scan was performed 2 weeks later to determine the location of pregnancy, the number of gestational sacs and fetal viability.
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Outcome measures
All analyses were performed using the Statistical Package of Social Sciences for Mac Version 22 (IBM Corp., USA). Continuous data were assessed for normality of distribution using the Shapiro–Wilk test, and expressed as mean (±SD) or median (interquartile range) accordingly; comparisons were made using the Student’s t-test and the Mann–Whitney U-test, respectively. Categorical data were expressed as number (percentage); comparisons were made using the chi-squared test or Fisher’s exact test and presented with rate ratios and 95% CIs. A two-sided value of P < 0.05 was considered as significant for all statistical tests performed.
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Results Participant flow The CONSORT diagram in Figure 1 demonstrates the participant flow. A total of 845 women were assessed for eligibility, of which 229 were recruited and ultimately 186 completed the study.
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Baseline and cycle characteristics The baseline and cycle characteristics of the ES and control groups are presented in Table 1. There were no statistically significant differences between the two groups for all the variables presented.
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The primary outcome measure was pregnancy rate, which was defined as a positive urine pregnancy test per embryo transfer. Secondary outcome measures were implantation rate, which was defined as the number of gestational sacs seen on ultrasound examination per embryo transferred; clinical pregnancy rate, which referred to the number of pregnancies with confirmed intrauterine gestational sac; ongoing pregnancy rate, which referred to the presence of at least one fetus with heart pulsation proceeding beyond 32 weeks of gestation; and live birth rate which referred to the number of deliveries with at least one live-born infant.
Outcome analysis The outcome analysis of the study is presented in Table 2. There was no significant difference in the pregnancy rate (ES group 48.4% versus control group 43.0%; RR 1.125, 95% CI 0.822–1.540) and the implantation rate (ES group 36.5% versus control group 32.8%; RR 1.143, 95% CI 0.802–1.627) between the two groups. In addition, there were no significant differences in the secondary outcome measures including clinical pregnancy and ongoing pregnancy or live birth rates between the two groups.
Please cite this article in press as: Jennifer Sze Man Mak, et al., The effect of endometrial scratch on natural-cycle cryopreserved embryo transfer outcomes: a randomized controlled study, Reproductive BioMedicine Online (2017), doi: 10.1016/j.rbmo.2017.04.004
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Figure 1 – CONSORT 2010 flow diagram.
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The findings were similar when performing intention-to-treat analysis with no statistically significant difference in the pregnancy rate (ES group 39.1% versus control group 35.1%; RR 1.115, 95% CI 0.795– 1.564) and the implantation rate (ES group 36.5% versus control group 32%; RR 1.143, 95% CI 0.802–1.627) between the two groups.
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Subgroup analysis and logistic regression
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Subgroup analysis was performed by stratifying women into those with one or more, two or more, or three or more previous ETs and two or more failed IVF attempts. No statistically significant differences were detected between ES and control groups in any of the outcome measures for any of the stratified groups (Figures 2 and 3).
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Discussion
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To the best of our knowledge this is the first RCT to evaluate the impact of ES on clinical outcomes in women undergoing natural-cycle FET.
We found no statistically significant differences between the two groups in the various clinical outcomes. Endometrial injury is defined as intentional damage to the endometrium performed to improve endometrial receptivity and therefore the implantation and pregnancy rates. We are aware that there are many methods to induce endometrial injury and there is no universal standard of practice. We know some investigators use a more invasive approach such as hysteroscopy-induced endometrial injury (Huang et al., 2011; Shokeir et al., 2016). However, most investigators use a more gentle approach using ES by pipelle (Nastri et al., 2015). We used this approach in this study, and our observation based on this soft ES showed that it does not have any beneficial effect on an unselected group of women undergoing FET in natural cycles. It is possible that the degree of injury may have an impact on pregnancy outcomes. However, there is no consensus on the optimal method of endometrial injury so as to exert maximal effect. Further adequately powered studies would be needed to clarify this. Although the recently updated Cochrane Review concluded a favourable effect of ES with an increased clinical pregnancy rate and live birth or ongoing pregnancy rate with moderate quality evidence
Please cite this article in press as: Jennifer Sze Man Mak, et al., The effect of endometrial scratch on natural-cycle cryopreserved embryo transfer outcomes: a randomized controlled study, Reproductive BioMedicine Online (2017), doi: 10.1016/j.rbmo.2017.04.004
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Table 1 – Comparison of baseline and cycle characteristics between the two randomized groups. Characteristics
ES group (n = 93)
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Baseline characteristics
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65 (69.9)
62 (66.7)
Tuboperitoneal, n (%)
45 (48.4)
46 (49.5)
Endometriosis, n (%)
16 (17.2)
21 (22.6)
Male, n (%)
42 (45.2)
44 (47.3)
5 (5.4)
3 (3.2)
infertilityc,d,#
1–2, n (%)
85 (91.4)
88 (94.6)
3–4, n (%)
8 (8.6)
5 (5.4)
1–2, n (%)
48 (51.6)
58 (62.4)
≥3, n (%)
24 (25.8)
20 (21.5)
1–2, n (%)
62 (66.7)
66 (70.1)
3–4, n (%)
5 (5.4)
2 (2.2)
1–2, n (%)
29 (31.2)
29 (31.2)
≥3, n (%)
8 (8.6)
9 (9.7)
Agonist
54 (58.1%)
61 (65.6%)
Antagonist
39 (41.9%)
32 (34.4%)
Baseline FSH (IU/l)b,*
7.3 (6.4–8.1)
7.1 (6.1–8.3)
Total gonadotrophin used
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(IU)b,
Oestradiol on trigger day (pmol/l)b,*
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Total oocytes retrievedb,*
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Insemination
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Intracytoplasmic sperm injection, n (%)
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Fertilization rate (%)a,‡
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Viable embryos for cryopreservationb,*
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Endometrial thickness (mm)a,‡
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Survival rate of warmed embryos (%)e
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*
10.92 ± 1.43
10.91 ± 1.56
3525 (2475–4500)
3300 (2250–4312)
7051 (3987–13057)
7824 (3750–13818)
11 (9–12)
10 (8–12)
44 (47.3)
43 (46.2)
49 (52.7)
50 (53.8)
methodb,#
Conventional, n (%)
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74.88 ± 18.01
73.16 ± 17.54
4 (3–6)
4 (3–6)
11.48 ± 2.30
11.13 ± 2.35
118/125 (94.4)
123/135 (91.1)
Number of embryos transferredc,#
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1, n (%)
71 (76.3)
64 (68.8)
2, n (%)
22 (23.7)
29 (31.2)
Day 2/3, n (%)
37 (39.8)
44 (47.3)
Day 5/6, n (%)
56 (60.2)
49 (52.7)
50 (43.5)
63 (51.6)
Stage of embryo transferredc,#
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Good-quality embryos transferreda, n (%)
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There were no statistically significant differences between the two groups. BMI = body mass index; ES = endometrial scratch; ET = embryo transfer. a
Data are mean ± SD; comparisons performed using Student’s t-test.
b
Data are median (25th to 75th centiles); comparisons performed using Mann–
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Whitney U-test.*
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Data are number (%); comparisons performed using chi-squared test# or Fisher’s exact test.†
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Some patients presented with more than one cause of infertility.
e
Number of embryos survived after warming/number of embryos warmed.
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Secondary, n (%)
Duration of stimulation (days)a,‡
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31 (33.3)
Treatment protocolc,#
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28 (30.1)
Cycle characteristics
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Primary, n (%)
Number of previous frozen ETc,#
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4.73 ± 3.26
Number of previous fresh ETc,†
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21.90 ± 2.81
4.74 ± 3.62
Number of previous ETc,#
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21.36 ± 2.81
)
Number of previous IVF attemptsc,#
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BMI
Unexplained, n (%)
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36.78 ± 3.45
Cause of
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36.90 ± 3.32
(kg/m2 a,‡
Type of infertilityc,#
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Age
(years)a,‡
Infertility duration (years)a,‡
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Non-ES group (n = 93)
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(Nastri et al., 2015), many confounding variables have been recognized, including the number of previous IVF failures, the number and quality of embryos transferred and the timing of ES. In addition, it is likely that another confounding variable is the type of embryo transfer cycle. Whilst the majority of earlier studies which demonstrated a beneficial effect of ES were based on fresh embryo transfer cycles (Barash et al., 2003; Guven et al., 2014; Inal et al., 2012; Karimzadeh et al., 2009; Mahran et al., 2016; Narvekar et al., 2010; Nastri et al., 2013; Raziel et al., 2007; Shohayeb and El-Khayat, 2012; Singh et al., 2015), there have been three studies examining the impact of ES in FET cycles (Aflatoonian et al., 2016; Dunne and Taylor, 2014; Kanazawa et al., 2017), each of which appears to have different results. In the first study by Dunne and Taylor (2014), which was a prospective cohort study examining 80 women with at least one failed IVF-ET, ES was performed between day 21 and 23 of the menstrual cycle preceding HRT-FET, and no statistically significant differences in implantation or clinical pregnancy rates were found between those with ES and those without. The second study was a randomized study by Aflatoonian et al. (2016) with 45 women in the ES group and 48 women in the Q8 control group; ES was performed between day 21 and 23 of the menstrual cycle preceding HRT-FET. However, the implantation and clinical pregnancy rates appeared to be lower in the ES group, but the difference was not statistically significant. The implantation rate was 11.8% versus 20.5%, clinical pregnancy rate was 22.2% versus 33.3% and ongoing pregnancy rate was 22.2% versus 31.2% in the ES and control groups, respectively. In a third study by Kanazawa et al. (2017), which was a retrospective cohort study examining a total of 173 women, in which patients mostly underwent HRT-FET (150 women on HRTFET and 23 on natural-cycle FET) and were divided into three groups: Group A (n = 38) underwent soft curettage to the endometrium twice prior to the FET cycle; Group B (n = 45) underwent hysteroscopy prior to the FET cycle, with no significant factors such as endometrial polyps; and Group C (n = 90) was the control group. The study found a positive effect of ES in FET cycles, reporting a statistically significant higher clinical pregnancy rate per transfer between Group A and Group C (42.1% versus 22.2%, adjusted odds ratios 2.49, 95% CI 1.01–6.17, P = 0.048), while Group B with only hysteroscopy had a higher pregnancy rate than Group C but showed no statistical significance (35.6% versus 22.2%, adjusted odds ratios 2.14, 95% CI 0.89–5.15). Considering the three studies together, it is impossible to draw a firm conclusion regarding whether or not ES in HRT-FET cycles is of benefit, especially because only one of the three studies was a randomized trial and one of them consisted of predominantly HRT cycles with only a small number of natural cycles, which may be another confounding variable. As far as we are aware, our study was the first conducted on the impact of ES on FET in natural cycles. We found no significant differences between the two groups in the various pregnancy outcomes. Whilst current data suggests that the implantation and clinical pregnancy rates are similar between FET in HRT cycles and natural cycles (Ghobara and Vandekerckhove, 2008; Groenewoud et al., 2016), it is unclear whether the impact of ES in these two types of cycle is the same. It is not scientifically sound to extrapolate data from one type of FET cycle to another. In this respect, a particular strength of our study was that it included only one type of FET cycle, namely FET in natural cycles, unlike the study by Kanazawa et al. (2017) which included a mixture of HRT (majority) and natural cycles. On the other hand, one possible criticism of our study, similar to that of Yeung et al. (2014), was the inclusion of patients with a variable number of previous IVF or embryo transfer attempts. It seems
Please cite this article in press as: Jennifer Sze Man Mak, et al., The effect of endometrial scratch on natural-cycle cryopreserved embryo transfer outcomes: a randomized controlled study, Reproductive BioMedicine Online (2017), doi: 10.1016/j.rbmo.2017.04.004
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Table 2 – Comparison of pregnancy outcomes per transfer between the two randomized groups (per protocol analysis). Parameter
ES group (n = 93)
Non-ES group (n = 93)
Relative risk 95% CI
Positive HCG ratea, n (%) Biochemical pregnancy ratea, n (%) Implantation ratea, n (%) Clinical pregnancy ratea, n (%) Miscarriage ratea, n (%) Multiple pregnancy ratea, n (%) Ongoing pregnancy/live birth ratea, n (%)
45/93 6/93 42/115 39/93 7/39 4/39 32/93
40/93 5/93 39/122 35/93 6/35 4/35 29/93
1.125 1.200 1.143 1.114 0.946 0.897 1.158
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(48.4) (6.5) (36.5) (41.9) (17.9) (10.3) (34.4)
(43.0) (5.4) (32.0) (37.6) (17.1) (11.4) (31.2)
(0.822–1.540) (0.379–3.795) (0.802–1.627) (0.782–1.588) (0.285–3.142) (0.242–3.321) (0.627–2.137)
ES = endometrial scratch; HCG = human chorionic gonadotrophin. a Data are number (%); comparisons performed using chi-squared test. There were no statistically significant differences between the two groups.
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that ES is more likely to have a beneficial effect in women with two or more ET failures compared with an unselected group of subjects undergoing IVF-ET (Nastri et al., 2015). Although it is possible to perform a subgroup analysis of subjects with regard to the number of previous failures, as in the case of our current study and that of Yeung et al. (2014), the subgroup analysis inevitably lacks sufficient power. In this respect, what we can conclude from this study is that ES does not appear to have any beneficial effect on an unselected group of women undergoing FET in natural cycles; but it is still uncertain whether or not ES has any benefit in women with recurrent implantation failure after IVF. Our power calculation was performed based on three previous meta-analyses in 2012, which reported at least a 50% increase in pregnancy rate after endometrial injury in the cycle prior to IVF (El-Toukhy et al., 2012; Nastri et al., 2012; Potdar et al., 2012). With the benefit of hindsight, the earlier estimate of an increase in pregnancy rate of FET from 30% to 50% following ES could well represent an overestimate. In order to remove bias as a double-blind RCT, we performed endocervical manipulation as control, on the assumption that cervical stimulation would not affect the outcome (if it did, it would be negligible because the procedure was confined to the cervical canal and should not disturb the endometrial environment). The exact mechanism by which ES may improve implantation remains unclear. Several possible mechanisms have been proposed (Dekel et al., 2010; Gnainsky et al., 2010; Kalma et al., 2009; Zhou et al., 2008). Whatever the underlying mechanism, it is highly plausible that the benefit, if any, of ES depends on the pre-existing condition of the endometrium. The current observation that ES is more likely to have a beneficial effect in women with recurrent implantation failure than an unselected group of subjects lends support to such a notion as women with RIF are considered to be more likely to exhibit abnormal endometrial features (Makrigiannakis et al., 2011). In this context, it is interesting to note that the implantation rate of fresh embryo transfer is generally considered to be inferior to vitrified-warmed embryo transfer because the endometrium in fresh embryo transfer cycles often exhibits dis-synchrony because of the supra-physiological levels of steroid hormones encountered in hyper-stimulated cycles (Bourgain and Devroey, 2003; Check et al., 1999; Devroey et al., 2004; Nikas et al., 1999; Ochsenkuhn et al., 2012; Richter et al., 2006; Roque, 2015; Roque et al., 2013, 2015; Shapiro et al., 2014), especially if the progesterone level on the day of HCG trigger is significantly elevated (Papanikolaou et al., 2012; Santos-Ribeiro et al., 2014; Venetis et al., 2013). It is tempting to speculate that ES is more likely to have beneficial effects in an abnormally developing endometrium (in stimulated cycles) than normally developing ones (in natural cycles).
Some investigators have speculated that ES may have adverse effects (Aflatoonian et al., 2016). Whilst we have found that ES did not have any beneficial effect, it did not seem to have any adverse impact either, with implantation and pregnancy rates very similar between those who did and did not have ES.
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Conclusion
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ES in the luteal phase of the cycle preceding the natural-cycle FET in an unselected group of women does not improve implantation and pregnancy rates.
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Acknowledgements
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The authors wish to thank research nurses Ms Eris Tse and Ms Cosy Cheung for helping with patient recruitment, randomization and data entry; Ms Pauline Chan for her valuable contribution to the data collection; Ms Mei-Chun Cheung, Ms Stella Tang and Ms Janet Lau in the ART unit for their help during the study. Finally, we are grateful to all the patients attending our unit for their willingness to participate in this study.
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A R T I C L E
I N F O
Article history: Received 2 December 2016 Received in revised form 4 April 2017 Accepted 5 April 2017
Declaration: The authors report no financial or commercial conflicts of interest.
Keywords: Endometrial injury Endometrial scratch Implantation Natural cycle Pregnancy rate Vitrified-warmed embryo transfer
Please cite this article in press as: Jennifer Sze Man Mak, et al., The effect of endometrial scratch on natural-cycle cryopreserved embryo transfer outcomes: a randomized controlled study, Reproductive BioMedicine Online (2017), doi: 10.1016/j.rbmo.2017.04.004
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Figure 2 – Subgroup analysis: Comparison of pregnancy outcomes between the two randomized groups with one or more, two or more and three or more previous embryo transfers. (A) One or more previous embryo transfers. There were no statistically significant differences between the two groups. (B) Two or more previous embryo transfers. There were no statistically significant differences between the two groups. (C) Three or more previous embryo transfers. There were no statistically significant differences between the two groups.
Please cite this article in press as: Jennifer Sze Man Mak, et al., The effect of endometrial scratch on natural-cycle cryopreserved embryo transfer outcomes: a randomized controlled study, Reproductive BioMedicine Online (2017), doi: 10.1016/j.rbmo.2017.04.004
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Figure 3 – Subgroup analysis: comparison of pregnancy outcomes between the two randomized groups with two or more failed IVF
Q10 attempts. There were no statistically significant differences between the two groups.
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