Live Birth after Cleavage-Stage versus Blastocyst-Stage Embryo Transfer in Assisted Reproductive Technology: A Randomised Controlled Study

Abstract

Background:

Blastocyst stage transfer appears to improve pregnancy outcomes. The aim of this study is to evaluate the pregnancy results between fresh cycle blastocyst stage embryo transfer and cleavage stage embryo transfer in patients who undergo intracytoplasmic sperm injection (ICSI).

Materials and Methods:

This randomised clinical trial study was conducted at the Infertility Research Centre of Milad Hospital in Mashhad, Iran from 2018 to 2020 on 240 infertile women who presented for their first ICSI procedure. These patients were assigned to receive either cleavage embryo transfer (n=112) or blastocyst stage transfer (n=107). Pregnancy outcomes were measured in both groups.

Results:

There were no differences regarding age, body mass index (BMI), serum follicle-stimulating hormone (FSH), duration of infertility, and aetiology of infertility between the groups (P>0.05). There were more follicles, total oocytes, and metaphase II (M2) oocytes in the blastocyst stage group. Considerably more cleavage stage embryos were transferred compared to the number of transferred blastocysts (P=0.001). The blastocyst group had more vitrified embryos than the cleavage group (P=0.000). The rates of implantation (P=0.332), chemical pregnancy (P=0.165), clinical pregnancy (P=0.694), and live births (P=0.727) were higher in the blastocyst group, but they were not significantly different. The rate of abortion was also not significantly higher in the blastocyst group (P=0.296).

Conclusion:

Blastocysts transferred in the fresh cycle of an ICSI procedure may be more advantageous compared to cleavage stage embryo transfer (registration number: IRCT20181030041503N1).

Introduction

The first successful birth following in vitro fertilization (IVF) was documented in 1978; since then, considerable efforts have been made to enhance its rate of success (1). For many years, embryo transfers were commonly conducted during the cleavage stage due to the absence of appropriate techniques and culture media for blastocyst transfer. However, advancements in sequential media have caused a shift towards blastocyst transfer on the fifth or sixth day after pick-up. Embryos transferred during the blastocyst stage have a higher rate of successful implantation (2-6) which is predominantly attributed to the enhanced capacity for evaluating embryo viability. This stage facilitates better coordination between the endometrium and the embryo and leads to a more successful implantation. Another advantage of blastocyst transfer is the ability to perform preimplantation genetic testing, when necessary (7- 9).

However, limitations of blastocyst transfer include a reduced proportion of embryos available for cryopreservation and increased transfer cancellation as a result of failure to achieve blastocyst formation (10- 14).

Papanikolaou et al. (13) assessed eight randomised controlled trials (RCT) and noted that the clinical pregnancy and live birth rates were considerably higher in blastocyst stage embryos compared to cleavage stage embryos when an equivalent number of fresh embryos were transferred.

A Cochrane Review conducted by Glujovsky et al. (15) examined 27 RCT and found that blastocyst transfer resulted in higher live birth and clinical pregnancy rates. However, there were no significant differences in terms of multiple pregnancies and miscarriages between the two groups. Subsequently, the aforementioned researchers conducted a Cochrane Review of 32 RCTs and reported that limited evidence exists for improved pregnancy outcomes in blastocyst transfer compared to cleavage embryo transfer. Although there are potential advantages of blastocyst transfer in fresh cycles, further data is required to determine the impact of the transfer stage on live birth and pregnancy rates. A recent systematic review of five RCTs that compared the effectiveness of single blastocyst stage embryo transfer with single cleavage stage embryo transfer indicated a higher rate of ongoing pregnancy and delivery in the blastocyst stage group. In light of these results, it was further proposed that larger and higher quality RCTs are imperative to determine conclusive results (16).

The advantages of a three-day or five-day embryo are a topic of ongoing debate (2). However, certain studies have demonstrated comparable pregnancy outcomes for IVF cycles after cleavage and blastocyst embryo transfers (8, 17, 18); in select instances, cleavage has been shown to be superior to blastocyst embryo transfers (17-20).

There appears to be a growing concern regarding the potential risk of epigenetic modifications that accompany a prolonged period of embryo culture (21-22). Therefore, the objective of this research is to undertake a comparative analysis of pregnancy outcomes between fresh blastocyst embryo transfer and cleavage embryo stage transfer in women who intend to undergo their first ICSI.

Materials and Methods

Study design and approval

This randomised clinical trial was conducted on a cohort of 250 women who referred to the In Vitro Fertilization Centre of Imam Reza Hospital (Mashhad, Iran) from 2018 to 2020. The study was approved by the Ethics Committee of Mashhad University of Medical Sciences (IR.MUMS.MEDICAL. REC.1397.617) and registered at the Iranian Registry of Clinical Trials (IRCT20181030041503N1). All participants signed an informed consent form for study participation.

Study participants

We anticipated that there would be a chemical pregnancy likelihood of 21.5% for cleavage embryos and 38.0% for blastocyst embryos based on our assessment of the available data and initial experiences during the study design phase. The sample size for this study with two independent groups was determined using G*Power software 3.1. Our required sample size for each group was 105 patients, for a total population of 210 women, after taking into consideration a one-tailed alpha error rate of 5% and a power of 80%. We assessed 250 patients after the determination of the sample dropout rate. From these, 10 patients were excluded-6 patients did not meet the inclusion criteria and 4 patients declined to participate. Therefore, a total of 240 patients enrolled in this study.

Patients who met the following criteria were included: i. 20-40 years of age with normal response to ovarian stimulation, ii. Normal uterine cavity, and iii. Presence of at least five embryos and sufficient endometrial thickness on day 12 of their cycle.

Patients were excluded if they had any of the following criteria: i. Lack of good quality embryos, ii. Presence of endocrine disorders (hypothyroidism, diabetes), iii. Poor responders with ≤3 oocytes in the previous cycle), iv. Excessive response to ovarian stimulation (>20 oocytes in the previous cycle), v. Only frozen embryos, or vi. History of uterine surgery, recurrent abortion, or diagnosis of severe male factor.

Randomisation

All study participants initially underwent a complete evaluation that included medical history, physical examination, and proper laboratory analyses. On the initial day of the embryo culture, the embryologist used a computer-generated randomised list to assign the patients to one of the two study groups. Those with an even number on the registration list were enrolled in the blastocyst transfer three-day group (n=120) and the remaining participants were assigned to the blastocyst transfer five-day group (n=120).

The Embryology Laboratory personnel contacted the patients to provide them with information regarding the scheduled day of embryo transfer as well as the number of fertilised embryos. The analyst and clinician were not informed about the participants’ study group assignments. Masking of group allocation was maintained until all procedures were completed and final data analyses were conducted.

Ovarian stimulation

We used three protocols for intracytoplasmic sperm injection (ICSI) after taking into consideration the age and diagnosis of the subjects.

Individuals enrolled in the long-acting gonadotropinreleasing hormone (GnRH) group received a daily subcutaneous dosage of 0. 1 mg GnRH analogue (Buserelin acetate, Germany) that began from the mid-luteal phase until the commencement of the subsequent menstrual cycle. The GnRH analogue dose was subsequently reduced to 0.05 mg/day until the day of ovulation triggering. Recombinant follicle-stimulating hormone (rFSH, CinnaGen, Iran) was prescribed after confirmation of pituitary suppression by ultrasound (no follicles >10 and endometrial line <5 mm).

In the short method, participants received Suprefact (Buserelin acetate, Germany) at a dosage of 50 mcg subcutaneously twice daily, commencing on the second day of the menstrual cycle and continuing until the induction of ovulation. rFSH administration commenced on the subsequent day. In the antagonist protocol, rFSH was administered on the second day of the menstrual cycle. Once the lead follicle attained a size of 12-14 mm, a daily dosage of 0.25 mg Cetrotide® (EMD Serono, Canada) was prescribed.

In all study participants, the initiation dose of gonadotropins was determined based on their age, previous cycle ovary response, ovarian reserve tests, and body mass index (BMI). The initial ovarian monitoring was conducted utilising ultrasound five days subsequent to the beginning of gonadotropin stimulation. The gonadotropin dose was adjusted based on the patient’s ovarian response as visualised by ultrasound. Human menopausal gonadotropins (hMG, Menotropin, Darou Pakhsh, Iran) was added when supplementation of luteinizing hormone (LH) was required. When three or more follicles achieved a diameter of 17 mm, 10 000IU of human chorionic gonadotropin (HCG, Pregnyl®, Darou Pakhsh, Iran) was administered. Ultrasound-guided transvaginal oocyte retrieval was conducted 36 hours after administration of the HCG injection.

During the morning of pickup, semen samples were collected and underwent density gradient centrifugation for the ICSI. The retrieved oocytes were preserved in cleavage medium (Origio, Denmark) under controlled conditions of 37°C, 6% CO₂ , and 5% O₂ . The cumulus cells that surrounded the oocytes were removed two hours after oocyte collection. Metaphase II (M2) oocytes were specifically chosen for ICSI. After undergoing artificial insemination, all embryos were subsequently cultured in sequential media.

Cytoplasmic injection of the sperm was performed and the evaluation of fertilisation was carried out after 16 to 18 hours. The presence of two pronuclear and two polar bodies indicated normal fertility. The embryos were assessed daily to evaluate their quality. A grade A embryo was defined to have four blastomeres on the second day and seven or eight blastomeres on the third day with an equal size and <20% fragmentation (high quality). Blastocyst embryos were assessed according to Gardner’s criteria (23).

One group received three-day-old embryos, while the other received five-day-old embryos for transfer, based on their randomisation assignment. All embryos were transferred using a Phillips transabdominal ultrasound (Affiniti 70) and a Cook catheter (Cook Medical, Australia). In the absence of blastocyst stage development, the most developed embryo was selected for transfer.

On the day of oocyte retrieval, patients received 600 mg of vaginal progesterone for 14 days. In the event of conception, vaginal progesterone was continued until weeks 8 to 10 of gestation.

Outcome measures

Serum β-hCG levels were assessed between 10- and 14-days post-transfer, and clinical pregnancy rates were determined by the observation of at least one foetal pole or pregnancy sac observed by transvaginal ultrasound at five weeks gestation. The fertilisation rate refers to the ratio of the total number of embryos to the number of M2 oocytes. The assessment of the implantation rate was undertaken by calculating the ratio of gestational sacs observed on transvaginal ultrasound to the total number of embryos transferred. Ongoing pregnancy was determined when the gestation period extended to 12 weeks or beyond.

Statistical analysis

The normal distribution of data was assessed using the Kolmogorov-Smirnov test. Descriptive data are expressed as mean ± SD and number (%). Demographic data were assessed by the student’s t test for independent samples and chi-square test. For quantitative data, the student t test was used. Qualitative data were analysed by the chisquare test. All tests were conducted with IBM SPSS Statistics 26.0 (STATSKEY.COM, USA). P<0.05 were considered to be statistically significant.

Results

Out of a total sample size of 250 patients assessed for eligibility, 10 individuals were excluded from the study due to their failure to meet the inclusion criteria as well as their refusal to participate. A total of 240 patients were randomly assigned to two groups, with 120 patients in each group. In the blastocyst group, a total of 107 individuals underwent the transfer procedure, while 13 individuals did not participate because of the absence of blastocysts (n=5), the presence of ovarian hyperstimulation syndrome (OHSS, n=3), and the presence of endometrial pathologies (n=5). In the cleavage stage group, 112 recipients underwent the transfer procedure, while 7 individuals did not (5 due to OHSS and 3 due to endometrial pathologies). Consequently, the final sample size for analysis consisted of 219 cases (Fig .1).

Fig 1.

Flow diagram. OHSS; Ovarian hyperstimulation syndrome.

Patient demographics and stimulation characteristics

Table 1 shows no significant disparities in terms of age, BMI, serum FSH levels, and duration of infertility within the two examined groups. The underlying cause of infertility was similar in both groups. There were significant variations observed between the groups regarding the type of stimulation protocol and the total dosage of gonadotrophin. The follicular count, the total count of oocytes, as well as M2 oocytes were found to be significantly higher in the blastocyst group compared to the cleavage stage group. A significantly greater proportion of embryos were transferred at the cleavage stage relative to the number of transferred blastocysts because of the reduced quantity of embryos that successfully progressed to the blastocyst stage in vitro in contrast to the number of embryos that reached the cleavage stage.

Table 1.

Patient demographics and stimulation characteristics

Characteristics	3-day	5-day	P value
Age (Y)	32.11 ± 5.813	30.72 ± 5.645	0.075
BMI (kg/m2)	25.086 ± 4.091	25.036 ± 3.937	0.927
Basal FSH (m IU/mL)	6.66 ± 3.03	6.24 ± 3.44	0.347
Type of infertility Primary Secondary	94 18	83 22	0.354*
Duration of infertility (Y)	5.59 ± 3.61	5.31 ± 3.572	0.570
Cause of infertility Male factor Female factor Unknown Female-male factor	37 (33.33)29 (26.12)33 (29.73)12 (10.81)	25 (23.58)31 (29.24)41 (38.68)9 (8.49)	0.312*
Stimulation protocol Long-term Microdose Antagonist	39 (34.82)33 (29.46)40 (35.72)	38 (35.51)16 (14.95)53 (49.54)	0.022*
Total gonadotrophin dose (IU)	2903.8636 ± 1119.188	2476.4019 ± 850.65	0.002
Trigger type HCG Deka Deka, HCG	99 (89.19)5 (4.50)7 (6.31)	83 (78.30)12 (11.32)11 (10.38)	0.079*
Endometrial thickness (mm)	8.64 ± 1.35	8.88 ± 1.33	0.188

Data are presented as mean ± SD or n (%). *; Chi-square test, BMI; Body mass index, FSH; Follicle-stimulating hormone, and HCG; Human chorionic gonadotropin. .

The blastocyst group had more vitrified embryos than the cleavage group (P=0.000). Furthermore, within the 3-day group, we observed that a single case presented with three gestational sacs and seven cases had two gestational sacs. On the other hand, in the 5-day group, four cases were observed with two gestational sacs.

Fertilisation and implantation rates

Table 2 shows that there are no significant differences between the two groups in both fertilisation and implantation rates, with P=0.527 and P=0.332, respectively.

Table 2.

Embryology data

Characteristics	3-day	5-day	P value
Number of samples	112	107	-
Number of gestational sacs (observed in sonography)	0.41 ± 0.653	0.41 ± 0.566	0.969
Follicles (number)	10.9286 ± 4.499	13.028 ± 4.239	0.000
Total oocytes (number)	7.47 ± 2.715	9.58 ± 3.561	0.000
M2 oocytes (number)	5.33 ± 2.466	7.44 ± 2.966	0.000
Embryos transferred (number)	1.86 ± 0.564	1.63 ± 0.523	0.001
vitrified embryos (number)	2.13 ± 2.152	4.70 ± 3.231	0.000
Fertilisation rate (%)	86.58	88.23	0.527
Implantation rate	37/111 (33.33)	40/107 (37.38)	0.332

Data are presented as mean ± SD or n (%). M2; Metaphase II.

Pregnancy outcomes

Table 3 shows no statistically significant differences in the chemical pregnancy and clinical pregnancy rates between the two groups, with P=0.165 and P=0.694, respectively. Within the first group, there were a total of eight instances of multiple pregnancies. Whereas, within the second group, there were four cases of multiple pregnancies.

Table 3.

Main outcomes

Characteristics	3-day	5-day	P value
Pregnancy (chemical)	32/112 (28.57)	40/107 (37.38)	0.165
Pregnancy (clinical)	29/112 (25.89)	30/107 (28.03)	0.694
Miscarriage	7/112 (7.14)	12/107 (11.21)	0.296
Live birth	25/112 (23.21)	27/107 (25.23)	0.727
Multiple pregnancy	8/111 (7.20)	4/107 (3.73)	0.262

Data are presented as n (%).

Miscarriage outcomes

Table 3 shows that there were no statistically significant differences in miscarriage rates between the two groups (P=0.296).

Live birth/pregnancy after 20 weeks

One case in the 5-day group had an ectopic pregnancy. The live birth rate observed in the cleavage groups was 23%, whereas the blastocyst group showed a slightly higher rate of 25%. The live birth rate in the cleavage groups was 23%, and the blastocyst groups exhibited a slightly higher rate of 25%. Nonetheless, no statistically significant difference in live birth rates was observed between the groups (P=0.727).

Discussion

The current study found that fresh blastocyst embryo transfer did not reveal any significant differences in terms of pregnancy outcomes in comparison to fresh cleavage embryo transfer. However, both chemical and clinical pregnancy rates were higher in the blastocyst embryo transfer group.

Advancements in culture media and laboratory protocols that include techniques for embryo selection (24, 25) and cryopreservation have significantly enhanced embryo viability (26, 27). As a result, there has been a consistent increase in blastocyst-stage embryo transfers at numerous infertility centres (28, 29). Approximately one-third of all transfers are conducted during the blastocyst stages (30). The benefits of blastocyst transfer in the assisted reproductive technology (ART) cycle are still a matter of debate. According to a 2022 Cochrane systematic review, more evidence is needed to understand if the embryo transfer stage has an impact on pregnancy outcomes (2).

We observed that the implantation rate was greater, but not statistically significant, in the blastocyst stage. The present study showed an increase in the rate of implantation in extended culture, which supported the findings of previous works (31, 32).

The results of our research are consistent with several published studies (17, 20, 33, 34). Alfaraj et al. (34) conducted a retrospective study and concluded that the transfer of 5-day embryos did not yield any additional advantage in terms of pregnancy outcomes compared to cleavage embryos. In line with our research findings, they indicated a significant elevation in the risk of miscarriage in blastocyst embryos (34). Hatırnaz and Kanat Pektaş (17) performed a prospective randomised trial that enrolled 190 women. The results demonstrated an increase in the implantation rate (47.7 vs. 45.3), clinical pregnancy (46.3 vs. 44.2), and live birth (36.8 vs. 34.7) for participants who underwent a 3-day transfer compared to those who underwent a 5-day transfer. The obtained results contrasted our findings, as the pregnancy outcomes were found to be higher in the group of embryos transferred after five days. However, it is important to note that despite these discrepancies, the differences between the two groups did not achieve statistical significance in either study. They concluded that the effectiveness of blastocysts transfer can be the same as cleavage embryo transfer. This finding is in contrast to previous studies where the results indicated a positive impact of blastocyst transfer on pregnancy rates (4, 35). Frattarelli et al. (36) conducted a RCT and demonstrated that blastocyst transfer yielded superior pregnancy and implantation rates in comparison to embryo transfer on day 3. The aforementioned findings were verified through a prospective randomised study conducted by Van der Auwera et al. (37). Several studies have examined the cumulative pregnancy rates following both fresh and frozen embryo transfer.

Fernández-Shaw et al. (7) showed a notable rise in cumulative pregnancy rates subsequent to a 5-day transfer as opposed to a 3-day transfer. The findings by De Croo et al. (38) yielded similar outcomes. Cameron et al. (39) conducted an assessment on the cumulative live birth rates during the initial complete cycle of IVF following both the cleavage-stage and blastocyst-stage transfers. Ultimately, their findings indicated that the utilisation of the blastocyst-stage transfer did not significantly impact the probability of achieving live birth during the first complete IVF cycle. However, cumulative clinical pregnancy rates that resulted from the cleavage stage exhibited equivalent or potentially superior outcomes when compared to blastocyst transfers. It is hypothesised that there has been a notable rise in the rate of the cancellation cycle and a significant decline in the number of embryos cryopreserved at the blastocyst stage.

The abortion rate observed in our study was higher in the blastocyst stage compared to the cleavage stage (7.14 vs.11.25). In contrast, several studies have documented a greater prevalence of miscarriage in 5-day embryo transfers as opposed to those conducted on the third day. Theoretically, there is an expectation that the abortion rate will diminish when the transfer of a superior-quality embryo is conducted within a synchronised uterine environment. However, a Cochrane review did not report a statistically significant difference in the occurrence of abortion when comparing the two transfer days. Therefore, it was concluded that there is uncertainty regarding the potential impact of blastocyst-stage transfer on the rate of miscarriage (2).

The incidence of multiple pregnancies observed in our study was determined to be 7.20% for 3-day embryo transfers and 3.73% for 5-day embryo transfers. It was anticipated that the incidence of multiple pregnancies would diminish with the utilisation of a 5-day embryo transfer approach, which is potentially attributable to a lower occurrence of aneuploid embryos at the blastocyst stage in comparison to the cleavage stage (40). However, according to a Cochrane review, it was found that 9% of women experience a multiple pregnancy following a fresh cleavage-stage transfer, while a range of 8 to 12% encounter this outcome after a fresh blastocyst-stage transfer. Among the subset of patients who underwent the transfer of an equivalent number of embryos, the utilisation of blastocyst transfer could potentially result in an elevation in the rate of multiple pregnancies (2).

A limitation in the present study was the small sample size. The lack of an adequate sample size results in statistically non-significant differences between the two study groups. Further research with a larger sample size should be undertaken to reach more reliable results. In addition, we did not evaluate the rate of cumulative pregnancy, which seems a more accurate marker to compare two different day transfers in the ART cycle. A strong point of this study is to perform an assessment of live births in both groups in another RCT.

Conclusion

Higher implantation, pregnancy rates, and live births were observed in patients who underwent blastocyst transfer despite the absence of statistically significant differences. It is imperative to conduct larger-scale data trials to investigate pregnancy outcomes. Also, it is necessary to report cumulative pregnancy rates that include fresh and frozen embryo transfers in future studies.

Authors’ Contributions

Mal.M., B.S.; Participated in the conception, Design, and Writing of the study. Mab.M.; Contributed to the data collection, Revision, and Editing of the study protocol. S.M.R.E., T.S.; Performed the data analysis and Interpretation. F.Z.; Collected the data and Wrote the first draft of this manuscript. All authors approved the final version of the manuscript for submission.