Tracking the implantation window: Synchronizing endometrial preparedness for implantation with stage of blastocyst to be transferred in antagonist IVF cycles involving single blastocyst transfers

INTRODUCTION

In nature, efficient reproduction relies on the synchronized behaviour of animals, the synchronized physiology of their reproductive organs and the synchronized interaction of the male and female gametes. This fundamental principle of synchronization has to be respected in assisted reproductive technology (ART) irrespective of the technique or species involved. Asynchrony and out of phase embryo transfer may lead to missing out the implantation window and unnecessarily hamper pregnancy rates.

It has been reported that increased uterine contractility during early luteal phase may adversely affect implantation potential of day 3 cleavage stage embryos.^[1,2] Conversely, the decline in this contractility towards the late luteal phase may enhance the probability of embryo implantation.^[3] This can simply be achieved by subjecting embryos to extended culture till day 5/6 post ovum pickup (OPU). With more stringent quality control measures in place and with the advent of better culture conditions using sequential media, day 5/6 blastocyst transfer (BT) indeed seemed to be a beneficial proposition for increased pregnancy rates. Several arguments have been offered in favour of extended culture.^[4] The most relevant being the observation that whereas not all top quality cleavage stage embryos form viable blastocysts, morphometric selection of the best looking day 5 blastocysts has considerably lowered aneuploidy rates. Blastocyst culture also enables transfer of fewer number of embryos per cycle and gives a better choice to opt for elective single embryo transfer (eSET), consequently resulting in reduced multiple gestation rates. An added advantage of blastocyst culture is that it offers adequate duration for gonadotrophin-induced raise in supraphysiological E2 levels to subside, thereby also providing ample time for improvement in endometrial receptivity (better temporal synchronization between embryo and endometrium during transfer) and eventually higher implantation rates.^[4]

Consequently, there were several studies suggesting not just BT but eSET to increase implantation rates.^[5,6,7] The trend of transferring expanded blastocysts continued till newer studies prompted assisted hatching, and yet another study^[8] reported better implantation rates with spontaneously hatching/hatched blastocysts. However, contradictory reports have kept pouring in. Whereas randomized trials in good prognosis patients revealed better implantation rates with BT compared to cleavage stage embryo transfer,^[9] subsequent trials in unselected patients revealed conflicting results. In addition, in unselected patients with previous one or more failed cycles, there was no difference in pregnancy and live birth rates (LBRs) with day 5 blastocyst or day 3 embryo transfer.^[10,11] Recent studies have also reported that BT does not improve the likelihood of pregnancy in poor prognosis patients.^[9] Attempts were then focused towards identification of ‘window’ period of implantation. ‘Implantation window’ is defined as the stipulated time during which the endometrium is rendered receptive for incoming embryo and outside of which time frame, it becomes non-receptive for implantation. Despite several studies focusing on the two most confounding factors, embryo development and endometrial receptivity, no substantial increase in take-home baby rates has been observed. The intricate mechanism of implantation still remains an enigma and ‘increased LBRs following in vitro fertilization (IVF)’ has been an Achilles’ heel for embryologists and clinicians alike!

We therefore tried to follow the basic premise that understanding the intricate correlation between embryo development and implantation window may be the key towards comprehending the implantation orchestra. The implantation window is generally believed to encompass days 17 to 24 of the menstrual cycle, during which period embryonic development and endometrial preparation for implantation occur in tandem. An embryo undergoes different stages of blastocyst development before it implants into a receptive endometrium. We contended that even a ‘top’-quality blastocyst transferred within the window period may fail to implant in an ultrasonographically detected healthy endometrium if it is not at the right stage of development on that particular day of period when transfer was performed. Therefore, our study sought to track the ‘implantation window’ by contemplating a relevant correlation between stage of blastocyst transferred and endometrial preparedness for implantation with respect to day of menstrual period in antagonist IVF cycles.

MATERIALS AND METHODS

Retrospective analysis of 443 cycles (February 2012 to September 2014) in eumenorrhic women with no previous implantation, undergoing IVF with antagonist treatment protocol using recombinant follicle stimulating hormone (FSH) for ovarian stimulation (Recagon 200 IU daily from day 2 to day of human chorionic gonadotropin (hCG) trigger). Regular ultrasonographic monitoring of the follicles was performed. When ≥2 follicles reached a size of 18-mm diameter, final trigger for oocyte maturation (injection hCG 5000 IU) was given. Oocyte retrieval was performed between 34 and 36 h after hCG trigger. Luteal phase support in the form of the micronized progesterone (Susten 100 mg daily) was provided. Oocyte/embryo donation and intra-cytoplasmic sperm injection (ICSI) cycles as well as freeze–thaw/vitrified–warmed embryo transfers were excluded to obviate any biases and to obtain a homogenous population of the women for study.

Blastocyst grading

Gardner’s system for blastocyst stage grading^[12] was followed, albeit with minor modification [Figure 1]:

• Stage 1: Early blastocyst − blastocele <50% volume of the embryo

• Stage 2: Full blastocyst − blastocele completely fills the embryo

• Stage 3: Expanded blastocyst − blastocele volume larger than that of early embryo accompanied by thinning of zona pellucida (ZP)

• Stage 4: Point of hatching blastocyst − trophectoderm starts herniating through ZP

• Stage 5: Hatching blastocyst: blastocyst has emerged out >25% from zona almost giving appearance of figure ‘8’

• Stage 6: Hatched blastocyst: blastocyst completely escapes from zona.

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Figure 1

Blastocysts: different stages and grades. Number represents stage of blastocyst, the first alphabet = grade of inner cell mass (ICM), second alphabet = grade of trophetodermal (TE) cells

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For blastocysts graded as 3 to 6, development of inner cell mass was assessed as follows:

1. tightly packed, many cells,

2. loosely grouped, several cells and

3. very few cells.

The trophectoderm was assessed as follows:

1. many cells forming a cohesive epithelium,

2. few cells forming a loose epithelium or

3. very few large cells.

RESULTS

Overall clinical pregnancy rate (CPR) was found to be 26.64% (118/443), whereas LBR was 21.67% (96/443). No cases of monozygotic twinning were observed. There was no statistically significant difference between the study group in the various IVF parameters [Table 1], suggesting that it was an unbiased homogenous study population. The levels of E2 (2097 vs. 2041 vs. 2316 vs. 2128 vs. 2140 vs. 2149 vs. 2093 vs. 2158 pg/ml; one-way ANOVA P = 0.75), luteinizing hormone (LH) (3.5 vs. 3.0 vs. 3.0 vs. 3.7 vs. 2.7 vs. 3.9 vs. 2.7 vs. 2.4 IU/l; one-way ANOVA P = 0.05) and progesterone (1.14 vs. 1.32 vs. 1.4 vs. 1.19 vs. 1.31 vs. 1.28 vs. 1.22 vs. 1.3 ng/ml; one-way ANOVA P = 0.57) on day of hCG also did not differ significantly among the groups based on transfers on day 17 through day 24, respectively. Similarly, the luteal phase levels of E2 (1074 vs. 927 vs. 1018 vs. 751 vs. 783 vs. 1057 vs. 755 vs. 631 pg/ml; one-way ANOVA P = 0.21) and progesterone (62 vs. 67 vs. 55 vs. 65 vs. 52 vs. 54 vs. 57 vs. 49 ng/ml; one-way ANOVA P = 0.11) on day 14 post sBT did not show any significant differences among the various groups. However, retrospective analysis revealed that LBR was influenced by transfer of various stages (1–6) of the blastocysts on different days of menstrual period (days 17–24, covering the implantation window) as depicted in Table 2. The likelihood of live birth is greatly enhanced with the transfer of the full blastocyst on day 17 (odds ratio: 11.18), Expanded blastocyst on days 18 to 20 (odds ratio: 1.8), blastocyst with zona breaker cells/point of hatching on days 21, 22 (odds ratio: 1.6) and hatching/fully hatched blastocyst on days 23, 24 (odds ratio: 2.83) compared to their transfers on any other day of the menstrual cycle. In our study, no clinical pregnancies or live births were obtained with the transfer of the early stage blastocyst on any of the days of the menstrual period. Expanded stage blastocysts on days 18 to 20; point of hatching blastocysts on days 21, 22; and hatching/hatched stage blastocysts on days 23, 24 correlated more strongly with live birth (Pearson r = 0.31, 0.41 and 0.75, respectively) than transfer of these stages of the blastocysts on any other days encompassing the implantation window period.

Table 1 IVF parameters in the study group during the implantation window period

Table 2 Live birth rates (LBR) in the study population during the implantation window period vis-a-vis stage of blastocyst transferred

DISCUSSION

Our results raise a pertinent question whether it is absolutely irrelevant to take into account the day of menstrual period while doing embryo transfer in IVF cycles? Debating the existence of the implantation window (∼days 18–22 of natural cycle) or considering day of the menstrual period may not hold much significance in IVF cycles involving day 3 cleavage stage embryo transfers. However, with an excellent blastocyst gradation system available and a rising trend towards day 5/6 BTs, it seems absolutely valid to investigate whether stage of the blastocyst transferred has any influence on CPRs if this transfer is synchronized with the day of the menstrual period. In addition, the gonadotrophin-induced raise in supraphysiological E2 levels is known to subside due to the extended culture.^[15] Most importantly, the temporal synchronization between embryo and endometrium almost mimics the physiological state owing to embryo culture to blastocyst stage.^[16,17]

This study provides a non-invasive insight into the so-called mythical implantation window and endometrial preparedness for implantation. It may be justified to mention that the implantation window can be rendered redundant and a mythical entity if the stage of the blastocyst transferred during this period is not taken into consideration. This study amply justifies the differential endometrial response to implantation observed during the implantation window period vis-à-vis stage of the blastocyst transferred. To our knowledge, no study has ever tried to correlate the days of the menstrual cycle during implantation window period with the stage of the blastocyst transferred.

In an attempt to enhance the overall take-home baby rates, recent efforts have focused on identifying clinical factors associated with blastocyst development and pregnancy. It has been believed that a non-multilayered endometrium consisting of homogeneous endometrial pattern (i.e. grade 4/IV) characterized by either hyper-echogenic or iso-echogenic endometrium is indicative of favourable endometrial response.^[18] However, because luteal phase support in the form of micronized progesterone was provided to all women and we transferred blastocysts only when endometrial thickness and echo pattern were in the favourable range, this ultrasonographic parameter seems redundant and further vindicates our point that the stage of the blastocyst probably triggers some endocrinological events in the endometrium which is of utmost importance for better receptivity and higher pregnancy rates. Latest research also proposes embryo glue^[19] and endometrial scratch^[20,21] as adjunct endometrial factors that may facilitate increased receptivity. However, trials are still underway and no conclusive evidence is yet available for the benefits of these techniques in improving LBRs in IVF cycles. Some studies have also suggested a role for serum 25-hydroxy-vitamin D levels in predicting implantation and CPRs,^[22] whereas its levels in follicular fluid have been reported to lower the possibility of achieving pregnancy.^[23] It would therefore be interesting to carry out a study combining any of these endometrial factors along with the considerations proposed in our present study.

CONCLUSION

The primary finding of our study is that as the day of the menstrual period advances, higher stage BT gives better pregnancy rates. This study emphasizes that synchrony between stage of the blastocyst transferred and the endometrial preparedness for implantation with respect to day of the menstrual cycle has a definitive influence on pregnancy rates in IVF cycles. Thus, the most practical application of these results is that if embryo development leads or lags with respect to endometrial receptivity, blastocysts may be frozen and transferred in the next conducive, coordinated natural cycle. The results may help establish a new paradigm for synchronized development of the embryo and endometrium for better pregnancy rates in IVF cycles.