The present study has demonstrated the good predictive potential of sub-endometrial VI index measurement on the maturation trigger day and LIF protein concentration in the implantation window for predicting endometrial receptivity that is characterized by successful implantation. Although the use of power Doppler indices as a marker has produced mixed findings , this study has displayed the favorable capacity of sub-endometrial VI index on the trigger day as an indicator for the likelihood of pregnancy. Considering the ever-changing process of sub-endometrial vascularization which is dynamically influenced by the estradiol and progesterone hormones [18, 19], serial assessments of the respective markers were obtained starting from the day of maturation trigger, ovum pick-up, and at the window of implantation of the fresh IVF cycle, while consecutive measurement in the frozen cycle was conducted by Nandi and Coworkers .
After performing multiple logistic regression analyses, our results were similar to the previous finding [2, 6] in regard to the optimal time point of sub-endometrial measurement. Ng and colleagues  demonstrated that the overall sub-endometrial indices assessed on the day of oocyte retrieval were not predictive of the pregnancy event. Likewise, a recent meta-analysis also failed to detect the significance of Doppler signal indices on the day of embryo transfer to differentiate women who achieved clinical pregnancy and women who did not . Interestingly, the present study confirms that the measurement of sub-endometrial VI on the day of maturation trigger could provide useful information among other time points in the fresh IVF cycle. Using different ultrasonography and Doppler parameters such as end-diastolic blood flow, endometrial pattern, and endometrial thickness, our results have supported the previous findings conducted by Dechaud and Colleagues , which established that the prominent information to predict the likelihood of pregnancy is attained when those parameters were measured on the day of trigger maturation rather than on the day of oocyte retrieval and embryo transfer.
On the contrary, a limited power of using sub-endometrial VI on the day of maturation trigger for pregnancy prediction was reported by Ng and Coworkers in another study . Nevertheless, the study design and the ovarian stimulation protocol were different from our study. While that study employed a long protocol for the IVF stimulation, our study recruited women who were subjected to the antagonist protocol. These differences might complicate the comparison of the study results. Another study also observed that the VFI index on the trigger day was highly predictive of pregnancy rather than the VI and FI indices which contradict our finding . On the other hand, endometrial VI, FI, and VFI were shown to be significantly higher in the pregnant group than those of the non-pregnant group [4, 5]. However, this study could not confirm those results since the areas of measurement were different.
The result of our study has also supported the expanding and consistent claims of LIF as a good marker to predict endometrial receptivity [8, 11, 12, 22]. Either glandular or luminal epithelial cells of the endometrium and also the blastocyst are known to express LIF and LIF receptors (LIF-R and gp130). Moreover, LIF expression in endometrium increases after ovulation day and attains its optimum concentration at the implantation window, suggesting the indispensable role of LIF and its receptors in the event of implantation. Simultaneously with a plethora of other cytokines, LIF is proposed to regulate the migration and invasion capacity of extra villous trophoblast on the maternal site. Once the syncytiotrophoblast has successfully invaded the luminal epithelial cells, the blastocyst starts to produce its own cytokines such as LIF and interleukin 1, which subsequently induce the increased expression of LIF in the endometrium. The crosstalk through cytokines is believed to be a remarkable mechanism that promotes the completion of implantation .
The successful implantation of LIF −/− mice blastocyst in the pseudo pregnant mice demonstrated the prominent role of the endometrial LIF rather than the embryo-secreted LIF. In humans, endometrial LIF expression level can be measured by utilizing different sampling methods including uterine secrete aspiration , flushing [11, 24], or biopsy [11, 25]. In a prospective follow-up study, Mikolajczyk and Coworkers  collected uterine flushing samples prior to laparoscopy for LIF measurements in infertile women diagnosed with minimal grade endometriosis (stage I-II), luteal phase defect, and idiopathic infertility. After eighteen months of receiving fertility treatments, questionnaires were sent out to all studied participants to follow up with the treatment outcomes. It was found that the infertile women who had failed to achieve natural pregnancy had a lower LIF expression than the women who had been pregnant (1.1 pg/mL Vs. 22.07 pg/mL; p value < 0.008). While the mentioned study suggested LIF as a predictor for the naturally conceived live-birth, our study has indicated its additional potential role in predicting the clinical pregnancy of an ovarian stimulation IVF program. Furthermore, the endometrial LIF expression presented in this study was closely related to the actual environmental condition of the uterus just before the embryo transfer procedure.
The strength of our study is in the presented results which suggest the availability of a reliable, quick, and simple alternative method to measure endometrial receptivity by simply aspirating the uterine secretion sample at the implantation window for clinical practice in the IVF program. We supported the results of previous studies that a small amount of endometrial secretion could be gently aspirated for the analysis of the various implantation biomarkers without reducing the chance of pregnancy [16, 17]. This current study has confirmed the safety and effectiveness of the endometrial secretion aspiration method for pregnancy prediction. Furthermore, as the gold standard of measuring endometrial receptivity relies immensely on the implantation event, by carefully selecting the good prognosis subjects who had successfully obtained good quality blastocyst for transfer, this study has reduced the potential bias that might arise from complications of the embryo.
This study also pertains to a limitation in which the number of participants is relatively small but, nonetheless, statistically adequate to prove the clinical use of sub-endometrial VI index and LIF as indicators for endometrial receptivity. Furthermore, this study has yet to discover the in-depth association between the VI index and the expression of LIF during the implantation window. Notably, a concordance decreased of VI index on the trigger day and low expression of LIF were more apparent in the non-pregnant group compared to the pregnant group. Further attempt to explore the relationship between VI index and LIF expression during the implantation window is worth pursuing in the interest of gaining a broader perspective on the pathophysiology of aberrant LIF secretion that leads to impaired implantation.
In conclusion, measuring the sub-endometrial VI index on the day of maturation trigger is most appropriate for a pregnancy prediction rather than on the day of oocyte retrieval and embryo transfer. Endometrial LIF expression at the window of implantation also poses as a potential predictor for pregnancy in IVF. A combination of the cut-off values of VI and LIF also yielded a simple noteworthy algorithm for the prediction of pregnancy.