The effect of embryo migration after embryo transfer with fresh oocyte donation cycles on pregnancy outcomes

Embryo migration is defined as the movement of embryos to implant at the exact site in the endometrial cavity during assisted reproductive technology (ART). We aimed to evaluate the impact of embryo migration on clinical pregnancy (CPR) and live birth rates (LBR) in fresh oocyte donation (OD) cycles. A total of 611 fresh OD cycles was recruited in this prospective cohort study. All embryos were expulsed to upper-middle uterus between 10 and 20 mm from the fundus. Air bubble-fundus distance was measured using ultrasound (USG) at the time of embryo transfer (ET) and then 60 minutes after ET. Patients were divided into 3 groups; first group consisted of patients whose embryos migrated towards fundus, second group whose embryos remained between 10 and 20 mm from fundus and the third group including embryos which migrated towards cervix. There was no significant difference between the groups regarding CPR and LBR (p = 0.359 and p = 0.865, respectively). Our study revealed that embryo migration was a fact and almost 22% of embryos migrated towards the fundus or the cervix. On the other hand, whether the embryo stayed static or migrated, CPR and LBR did not differ significantly in fresh OD cycles.

Success of assisted reproductive technologies (ART) which is expressed by means of clinical pregnancy rate (CPR) and live birth rate (LBR) depends on embryo quality, endometrial receptivity and embryo transfer (ET) technique (Buckett, Citation2006; Mansour & Aboulghar, Citation2002). Embryo transfer is the final crucial step to affect success rates and to date there has been vast number of studies to optimize ET technique for better implantation and increasing CPR and LBR. The effect of ultrasound (USG) guidance, removal of cervical mucus prior to ET, difficulty of transfer, catheter type, blood or mucus presence on catheter, embryo injection speed, time between loading of embryo and injection to uterine cavity were analyzed in various studies (Arora & Mishra, Citation2018; Eskandar et al., Citation2007; Groeneveld et al., Citation2012; Matorras et al., Citation2004; Moragianni et al., Citation2010; Spitzer et al., Citation2012). Another important issue is where the embryo is expulsed in the uterine cavity during transfer. After extensive use of USG guidance, authors examined the effect of the distance between the catheter tip and uterine fundus on CPR. A randomized controlled trial in literature recommended to expulse embryos in upper middle part of the uterine cavity to avoid fundal contact and not to induce uterine contractions (Oliveira et al., Citation2004). Recently, the American Society for Reproductive Medicine (ASRM) published a guideline about how to perform ET to improve the pregnancy outcomes. They recommended to place ET catheter tip in the upper or middle area of uterine cavity and expulsing the embryo more than 10 mm from the fundus under USG guidance. It was also recommended to use soft transfer catheters to improve CPR and LBR (Practice Committee of the American Society for Reproductive Medicine, Citation2017). Embryos are loaded on the catheter mostly in the order of air-embryo-air-medium. By doing so, we indirectly visualize embryo(s) via air bubbles and we rely on bubble position as a reflector of embryo position (Soares et al., Citation2008). For a long time, it was thought that the embryo did not move in the uterine cavity and remained at initial expulsion site. However, Edwards and Steptoe reported a case report presenting tubal ectopic pregnancy after in vitro fertilization (IVF) with a day-3 ET in 1976, which may be accepted as the first evidence for migration of embryos from the uterine cavity (Steptoe & Edwards, Citation1976). Then the migration concept has been taken into consideration by the authors performing investigations on IVF treatments (Fıçıcıoğlu et al., Citation2018; Ozdemir et al., Citation2019; Saravelos et al., Citation2016). Oocyte donation (OD) is a method of ART in which fertilization is achieved using donor oocytes to optimize the embryo quality (Kaser et al., Citation2019). Oocyte donation cycles which are the choice of treatment in certain indications such as advanced reproductive age (>40), repeated IVF failure, premature ovarian insufficiency (POI) and particular heritable genetic diseases have high LBR (Weissmann-Brenner et al., Citation2015). Donor oocytes can be used as fresh or cryoprotected-thawed although fresh ones have been found to have higher LBR (Centers for Disease Control & Prevention, Citation2017). In literature, there are no studies performed with fresh OD cycles about effect of air bubble-fundus distance after ET, and there are no studies performed whether there is any embryo migration after 60 minutes of ET and its pregnancy outcomes. We conducted this study to evaluate the impact of embryo migration on CPR and LBR in fresh OD cycles following USG-guided ET.

This study was conducted at an international IVF Centre between January 2016 and May 2020. All procedures were routine standardized procedures applied in the centre. The authors assert that all procedures contributing to this work comply with the ethical standards of the relevant national and institutional committees on human experimentation and with the Helsinki Declaration of 1975, as revised in 2008. In addition, the authors assert that all procedures contributing to this work comply with the ethical standards of the relevant national and institutional guides on the care and use of laboratory animals. The study was approved by Yakin Dogu University Ethical Committee (YDU/2020/86-1239). The study was registered to clinical trials (NCT044855669). The eligible patients were asked for oral and written consents for their data to be included in the analysis. The study included fresh OD cycles of recipient women having anteverted-anteflexed uterus anatomically. Frozen-thawed embryo transfer cycles, cryopreserved-thawed OD cycles were excluded. Partners with normal sperm analysis were included whereas those with abnormal sperm parameters or need of any testicular interventions were excluded from the study. Cases with difficulty during transfer were also excluded from the study. All treatment procedures including ovarian stimulation, oocyte retrieval and ET were performed by the same attending physicians (AE and BA). Author TG, HGC, JY and EB did not participate in the recruitment or clinical care of oocyte donors or their recipients. Age, body mass index (BMI), indication for OD, the number of metaphase II (MII) oocyte and day-5 embryo, quality of embryos, embryo number per transfer, endometrial thickness measured on ET day, air bubble-fundus distance measured by transabdominal USG at 1st minute and 60th minutes following ET, presence of blood or mucus on catheter tip and pregnancy outcomes were recorded. In current study, fresh OD recipients were divided into 3 groups according to change of embryo location between 1st and 60th minutes after ET. Embryos moved nearer to uterine fundus in Group 1, embryos were found in same expulsed zone in Group 2 and embryos moved further away from initial expulsed zone towards cervix in Group 3. Donor preparation for fresh OD Reproductive-aged oocyte donors did not have any contraindication for donation including any sexually transmitted diseases and genetic abnormalities. Donor cycles were prepared with flexible progestin primed ovarian stimulation protocol (Yildiz et al., Citation2019). For all recipients, at least 12 fresh MII donor oocytes were used. Fertilization was performed by experienced embryologist with intracytoplasmic sperm injection (ICSI). All sperms were selected via swim-up procedure before ICSI (Gardner et al., Citation2000). All embryos were monitored with time-lapse system (EmbryoScope+, Vitrolife). Fertilization was evaluated at 18–20 hours after ICSI whereas morphologic scores of embryos were assessed on day-3 and day-5 of ET. All transferred embryos were at blastocyst stage and grades were as 5AA, 5AB, 5BA, 4AA, 4AB or 4BA (Alpha Scientists in Reproductive Medicine & ESHRE Special Interest Group of Embryology, Citation2011). Endometrium preparation Endometrial preparation in recipient women was performed by one of two methods; (i) postmenopausal women received cyclic oestrogen-progesterone (Cyclo-Progynova, Bayer) for 2 months, (ii) perimenopausal and premenopausal women received single dose of 3.75 mg leuprolide acetate injection for pituitary down-regulation. On 2nd or 3rd day of following menstruation, all recipients were evaluated with transabdominal USG (DC 60 Exp 2018, Mindray, China). Following the presence of any cystic mass on ovaries was ruled out and thin regular endometrial lining was confirmed, 6 mg daily oestrogen (Estrofem, Novo Nordisk) was initiated. 7–10 Days later, endometrium was evaluated again with transabdominal USG. If the endometrial thickness was less than 7 mm or not trilaminar in pattern, the oestrogen dose was increased to 8 mg daily and continued for additional 7 days. When the endometrial thickness was less than 7 mm despite using 14–17 days of oestrogen or irregular pattern was observed, those individuals were excluded from the study. Transdermal or vaginal oestrogens were not used. When oocyte pick-up (OPU) procedure was applied to the donor, the recipient received 1 × 1 subcutaneous progesterone (Prolutex 25 mg, IBSA) simultaneously for luteal phase support. Six days after progesterone injection, blastocysts were evaluated and embryo transfer was performed. Embryo transfer technique All embryo transfers were performed in a standardized fashion by same experienced reproductive endocrinologist (BA) in our centre. During ET procedure, after application of speculum, G-rinse medium (Vitrolife) was used to remove any mucus on external cervical ostium, sterile gauze was also used if needed. Embryos were loaded to Wallace catheter (Soft 23 cm, Classic, 1816 N, CooperSurgical, Inc.) with air-embryo-air-medium order. Total volume to be transferred was restricted to be 0.2 mL. The air bubble seen on transabdominal USG at the time of ET refers to the embryo(s), which is an indirect non-invasive marker of embryo. One or 2 embryos were transferred per recipient. All ET was neatly performed via transabdominal USG guidance. The embryos were expulsed in upper middle uterus between 10 and 20 mm from the fundus via the soft catheter, avoiding touching the fundus. The catheter was examined for any retained embryos immediately. The patient was evaluated with transabdominal USG for the distance of air-bubble from uterine fundus at the 1st and 60th minutes after ET. Embryo fundal distance measurement was performed at the time of ET. Embryo was expulsed to the upper middle uterus between 10 and 20 mm from the fundus. The distance between uterine fundus and air bubble (which was assumed as an indirect noninvasive marker of embryo) seen on trans-abdominal USG imaging immediately at the time of ET, was accepted as embryo fundal distance in millimeters. The fresh oocyte recipients were divided in three groups according to embryo migration at 60th minute comparing with the initial expulsed zone. The Group 1 consisted of patients whose embryos migrated towards fundus, Group 2 had embryos remained between 10 and 20 mm from fundus and Group 3 had embryos migrated towards cervix. The groups were compared for pregnancy outcomes including CPR and LBR. Evaluation of pregnancy Beta human chorionic gonadotropin (beta-hCG) levels were measured 11 days after ET. Beta-hCG >50 IU/ml was accepted as ‘pregnancy’ whereas <50 IU/ml was not pregnant. Beta-hCG measurement was repeated 48 hours later and if the level was decreasing, it was termed as ‘biochemical pregnancy’. Presence of intrauterine gestational sac with fetal cardiac activity 5 weeks after ET was accepted as ‘clinical pregnancy’ (CP). Any pregnancy located outside uterine cavity was named as ‘ectopic pregnancy’. The intrauterine pregnancy was named as ‘miscarriage’ when terminated before 20 weeks of gestation. Any pregnancy terminated beyond 20 weeks was termed as ‘birth’. The birth of a live fetus was named as ‘live birth’ (LB). CP per ET was named as CPR and LB per ET was named as LBR (Barnhart, Citation2014). Statistical analysis Statistical analysis was performed using the SPSS v.22 (Statistical Program for Social Sciences, IBM, Chicago, IL, USA). Data was characterized by means, standard deviations (SD) and percentages. Kolmogorov-Smirnov Shapiro-Wilk test was used for assessing normality. Parametric tests were used for variables fitted to normal distribution, and non-parametric tests used for not normally distributing variables. Two independent t-tests were used to compare parametric variables. One-way analysis of variance (ANOVA) with Bonferroni post-hoc test was used for comparing continuous variables. Non-parametric three groups were compared with independent samples Kruskal Wallis Test when significant pairwise comparison of groups was done with Mann Whitney-U test. Categorical variables were compared with χ2 test. A p value <0.05 was considered as statistically significant.

A total of 653 fresh OD cycles who met the eligibility criteria were included in this prospective cohort study. Of those, 42 women were excluded from the study as difficult ET was experienced in 31 patients and the partners of 12 patients had abnormal spermiogram or required testicular intervention. Remaining 611 fresh OD cycles were further analysed. The mean age of the participants was 41.5 ± 5.6 while the mean BMI was 24.1 ± 3.3 kg/m2. The indications of OD were advanced maternal age in 438 women (71.7%), recurrent IVF failure in 103 women (16.8%), POI in 44 women (7.2%) women and hereditary or genetic diseases in 26 women (4.3%). The mean number of MII oocytes and day-5 embryos were 13.0 ± 1.5 and 5.1 ± 2.3, respectively. The mean endometrial thickness was 9.0 ± 1.9 mm. A total of 1069 embryos were transferred following the assessment of their quality. Of those, two embryos were transferred in 458 patients while one embryo was transferred in 153 patients. Because all difficult transfer procedures were excluded from the study, there was no blood or mucus on the catheter tip in any transfer. The mean air bubble-fundus distance was 13.2 ± 1.8 mm and 12.5 ± 3.1 mm at 1st minute and 60th minute after ET, respectively. Group 1 included 123 patients (20.1%) who had embryos located closer than 10 mm to the fundus, embryos were located at same initial expulsed zone between 10 and 20 mm from the fundus in 476 patients (77.9%) (Group 2) and embryos moved more away from fundus towards cervix in 12 patients (2%) (Group 3). There was no significant difference in terms of age, BMI, mean number of day-5 embryos and number of transferred embryos and air bubble-fundus distance at 1st minute after ET between these three groups (). Air bubble-fundus distance measured at 60th minutes of ET was significantly different (p < 0.001 between Group 1 and 2, p < 0.001 between Group 2 and 3, p < 0.001 between Group 1 and 3). Endometrial thickness was also found to be different significantly (p < 0.001 between Group 1 and Group 2, p < 0.001 between Group 1 and 3). There was no significant difference regarding endometrial thickness between Group 2 and 3 (p = 0.07). There were 447 clinical pregnancies in 611 fresh OD cycles (73.2%). Of those, 400 pregnancies resulted in live births (65.5%) and 47 patients (7.7%) had miscarriage. There were 96 clinical pregnancies (78.0%) in Group 1; 63.4% of them (n = 78) had live birth and 18 (14.6%) ended with miscarriage. There were 343 clinical pregnancies (72.1%) in Group 2; 314 had live birth (66%), 29 (6.1%) ended with miscarriage and there were 3 ectopic pregnancies (0.6%). There were 8 clinical pregnancies (66.7%) in Group 3; all resulted in live birth (66.7%). The groups did not differ significantly regarding CPR. We did not find any significant difference in terms of LBR among three groups (). Miscarriage rate was significantly different only between Group 1 and 2 (p < 0.001). The embryo which most moved towards fundus was initially located at 17.7 mm at 1st minute and it was measured to be at 6.1 mm from fundus at 60th minute. There was 11.6 mm replacement contributing to 65.5% change. This patient ended up with live birth eventually. The embryo which most moved towards cervix was initially located at 10.6 mm at 1st minute and it was found to be at 23.3 mm from fundus at 60th minute. There was 12.7 mm replacement towards cervix contributing to 119.35% change. This patient had no clinical pregnancy. presents the effect of embryo movement on pregnancy outcomes. The percentage of embryo movement had no significant impact on CPR or LBR.

Embryo transfer technique is one of the important steps during IVF treatments to improve CPR. The parameters such as embryo expulsion rate, clinician’s experience, type of the catheter, pressure used to press the plunger and intrauterine resistance have been considered to have an effect on the pregnancy outcomes (Fıçıcıoğlu et al., Citation2018). Embryo migration is a rather new concept in the literature. To the best of our knowledge, there is no study searching the association between embryo migration and LBR in fresh OD cycles and our study is the first to enlighten this topic in a large population. In our study, we aimed to assess whether there is any impact of embryo migration on CPR and LBR in fresh OD cycles. We found that CPR and LBR were not significantly affected regardless of embryo movement or direction of movement. All transfers were performed gently by the same experienced reproductive endocrinologist, at a constant injection speed, with constant 0.2 mL of fluid loaded to a soft catheter, without traumatizing endometrium and meanwhile avoiding touching the fundus to maximize the success rate. There are some detrimental effects of hormonal treatments used during ovarian hyperstimulation on endometrium in fresh autologous IVF cycles (Oliveira et al., Citation2004; Shaia et al., Citation2020). On the other hand, artificially induced endometrium can result in adequate preparation for embryo implantation without compromising the pregnancy outcomes in frozen-thawed ET and fresh OD cycles (Casper, Citation2020). Embryos prefer to implant most commonly in the upper and middle-posterior sites of endometrium in pregnancies following spontaneous conception (Minami et al., Citation2003; Nikas et al., Citation1995). There are papers evaluating the association between embryo expulsion sites and pregnancy outcomes, some of which detected increased CPR with lower positioning of the catheter tip in the uterine cavity (Coroleu et al., Citation2002; Frankfurter et al., Citation2004) while others found increased CPR with central positioning of the catheter tip (Oliveira et al., Citation2004; Tiras et al., Citation2010) and no relationship was found between position of catheter tip and CPR (Franco et al., Citation2004). The ASRM guideline recommends to expulse embryos at the upper-middle portion with a distance of >10 mm from the fundus (Practice Committee of the American Society for Reproductive Medicine, Citation2017). In our study, we expulsed all embryos to the upper-middle area of the cavity as recommended in the guideline. In accordance with the literature, our results demonstrated that CPR and LBR were 73.2 and 65.5% in 611 fresh OD cycles, respectively (Kushnir et al., Citation2018). Saravelos et al. (Citation2020) visualized the embryo at the upper part of the uterine cavity via a 3-dimentional USG soon after ET and they demonstrated four ectopic pregnancies occurring, which also supported the concept of embryo migration. There were tubal, cervical, interstitial and ovarian ectopic pregnancies reflecting embryo migration (Saravelos et al., Citation2020). In their study, Baba et al. found 81% of embryos to stay at initial expulsed site and Woolcott and Stanger found 94.1% of embryos did not move after ET (Baba et al., Citation2000; Woolcott & Stanger, Citation1998). On the other hand, most embryos were found to have moved from their initial expulsion site after ET in a recent study, most of them moved towards uterine fundus (Saravelos et al., Citation2016). The most predictive time for clinical outcome has been found to be 60 minutes after ET rather than 1 or 5 minutes after ET (Saravelos et al., Citation2016). In our study, we checked air bubbles 60 minutes after ET and we found that 77.9% of embryos stayed ‘static’ in their initial expulsed site. In contrast to our results, there are studies revealing lower pregnancy rates with migration of embryos towards cervix (Fıçıcıoğlu et al., Citation2018; Saravelos et al., Citation2016; Tiras et al., Citation2012). Fıçıcıoğlu et al. analyzed 230 fresh IVF cycles for embryo migration after 60 minutes of ET. They found that CPR was higher in the group having embryos with movement towards fundus at 60 minutes (Fıçıcıoğlu et al., Citation2018). Ozdemir et al. compared embryo flash migration in fresh and frozen ET and they found no significant difference regarding pregnancy outcomes between patients with and without embryo migration in frozen transfers. However, pregnancy rate was lower in patients with embryos having cervical displacement in fresh transfers (Ozdemir et al., Citation2019). In our study, there were 12 patients with embryos moving towards cervix and CPR and LBR were 66.7 and 66.7%, respectively. Our study showed that cervical movement had higher CPR compared to the current knowledge in the literature. The increased pregnancy rate could be explained with the use of good quality embryos and young donor oocytes who had better implantation capacity. In our study, endometrium thickness of Group 3 was significantly thicker than Group 1. Endometrium thickness may also have a positive association with implantation of embryo in the uterine cavity. The uterus may have some degree of contractions that move embryo(s) towards the implantation site. The ET procedure itself can initiate uterine contractility (Schoolcraft et al., Citation2001). Saravelos and Li stated that the contractions could change the embryo position away from initial expulsed site and implantation can occur several hours or days later (Saravelos & Li, Citation2019). Migration of expulsed embryos are hypothesized to be the result of gravity or uterine contractility in a study by Zhu et al., Citation2014. We included only patients with antevert anteflexed uteri to overcome any gravitational effect on our population. Anteverted anteflexed uteri lie parallel on bladder and we can state no or minimal effect of gravity as a factor for embryo replacement. Our study has several strengths. This is the first study to search the impact of embryo migration on LBR in fresh OD cycles in a large sample size. Day-5 embryos were transferred in all participants who were followed for a long period getting information about their pregnancy and live birth results. Another positive aspect of our study is having all patients receiving good quality embryos via OD cycles. Contrary to fresh autologous IVF cycles, due to bypassing the negative effect of ovarian hyperstimulation agents on endometrium, OD cycles are free of this negative effect. Fresh OD cycles have another advantage over frozen OD cycles, as oocytes are not exposed to potential harmful effects of cryoprotectants, freezing or thawing procedures. Although our population had sufficient number of participants with analyzing LBR, further prospective studies are required to confirm the effect of embryo migration on pregnancy outcomes. As a conclusion, our study reveals that the concept of embryo migration is a fact and almost 20% of embryos migrate towards fundus or cervix. On the other hand, whether embryo stayed static or migrated, CPR and LBR are not affected from any possible migration.

The authors would like to thank the participants of this study. Disclosure statement No potential conflict of interest was reported by the author(s).

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