The Immune Milieu After Local Endometrial Injury in Women with Recurrent Implantation Failure

The Immune Milieu After Local Endometrial Injury in Women with Recurrent Implantation Failure | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article The Immune Milieu After Local Endometrial Injury in Women with Recurrent Implantation Failure Yiping Fan, Ryan Wai Kheong Lee, Archita Mishra, Tse Yeun Tan, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7494176/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 30 Dec, 2025 Read the published version in Scientific Reports → Version 1 posted 11 You are reading this latest preprint version Abstract Recurrent implantation failure (RIF) occurs in 10–15% of IVF cycles with evidence from a few randomized control trials (RCTs) that local endometrial injury (LEI) leads to higher live birth rates whose exact mechanism is currently unknown. During the implantation period, modulation in immune milieu occur in tandem with profound morphologic and functional changes in the endometrium. The landscape of immune cells in the endometrium in pre- and post-LEI in RIF is currently unknown. Thirty-seven women with RIF (age 34.6 ± 3.3 years old) underwent LEI by two sequential mid-luteal phase endometrial biopsies prior to embryo transfer. To characterize the immunological landscape alterations in LEI, we performed immunophenotypic assessment with flow cytometry to provide insights into the basal (first biopsy) and altered (second biopsy) biology of dendritic cells (DC), macrophages, natural killer (NK), T and B cells in the RIF population before and after LEI. Clinical pregnancies occurred in seventeen women (46%). Among analysed immune cells, T (34.6%) and NK cells (26.2%) predominate in the mid-luteal endometrium. A consistent increase in lymphocytes and decrease in antigen presenting cells (APCs) were observed between the two biopsies although not statistically significant. Segregating by pregnancy outcomes demonstrated a significant increase in the T cells in the women who did not get pregnant post the local endometrial injury which was not observed in the group of women with RIF who fell pregnant (p = 0.03). There were no further difference in any of the other measured immune cell subsets between the first and second endometrial biopsy. We found limited changes in the immune cell compartments after LEI. Further research with higher resolution methods may provide more information on the effects of LEI. Health sciences/Diseases Biological sciences/Immunology Health sciences/Medical research Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Advancements in reproductive techniques have improved the live birth rates of in-vitro fertilization (IVF) cycles. However, live-birth rates of IVF have plateaued around 30% 1 for over a decade despite extensive research into embryonic factors and endometrial receptivity 2 – 4 . Embryo implantation 5 remains the rate limiting step in IVF especially in a small group of women with recurrent implantation failure (RIF). The definition of RIF is currently not universally defined. Proposed RIF definitions include failure to achieve a clinical pregnancy after transferring at least four good quality embryos following two to six embryo-transfers (ET) 6 – 9 . It was previously observed that local endometrial injury (LEI) by endometrial scratching before IVF led to a two-fold increase in pregnancy rates 10 , 11 . Endometrial scratch involves obtaining tissue biopsy using a sampler such as a Pipelle catheter where a ‘scratch’ is performed that is reasonably well tolerated without any need for analgesia 12 . Since then, there have been many randomized clinical trials evaluating the impact of LEI on pregnancy rates prior to IVF 13 – 16 . In the presence of inconclusive and contrasting evidence, there is no current consensus whether patients should receive endometrial scratching prior to IVF 17 . The majority of randomized controlled trials (RCTs) showed a general trend towards benefit of LEI especially where the number of previous failures are more than two. In order to improve the power to detect possible differences, meta-analyses conducted by both Vitagliano et al and van Hoogenhuijze et al both demonstrated improved live-birth rates post LEI in women with multiple failures 18 , 19 . One RCT that stood out was the PIP trial that did not show a benefit in a subgroup of women with two or more failures 20 . However, the PIP trial was designed to test the effect of LEI in a unselected group of patients and not women with RIF. The most recent large RCT, SCRaTCH trial, showed a trend towards benefit after one IVF cycle failure and with a single luteal phase LEI 21 . A more updated meta-analyses provided by Vitagliano et al that included both the PIP and SCRaTCH trial continued to show a benefit of Live Births (RR 1.21, 95CI 1.05–1.40) 21 . The timing, frequency and type of LEI instituted varied considerably between the various studies, making it difficult to generalize the findings 14 , 19 , 22 . It is therefore important and timely to define the type of patients being offered this intervention, the way LEI is administered in this specific group of patients with RIF to maximize their chances of getting pregnant. Putative mechanisms on how LEI may improve implantation rates in patients with RIF are currently unknown. Some proposed mechanisms include increasing endometrial receptivity by altering decidualization favouring implantation 23 , provoking the endometrial immune system to generate an inflammatory response 24 leading to increased secreted cytokines, growth factors and upregulation of adhesion molecules 24 – 26 . The feto-maternal immune cross talk is complex with recruitment and modification of endometrial immune cells involved in implantation 27 . Research into the immune milieu in the endometrium have shown that the roles of decidual immune cells are associated closely with implantation, angiogenesis and maintenance of pregnancy 28 , 29 . These include macrophages, dendritic cells (DCs), natural killer (NK) cells, lymphocytes such as B cells and T cells which are involved with implantation and feto-maternal tolerance towards pregnancy 5 , 30 , 31 . We have previously studied the effect of LEI on SUSD2 + mesenchymal stromal cells using a sequential mid-luteal Pipelle approach 23 . However, the effects on the various subsets of immune and APCs in subsequent menstrual cycles are unknown. To date, there has been no properly performed study that characterizes tissue resident immune cell sub-sets before and after LEI in subsequent luteal phase in women with RIF, and how any alterations in immune cell types and compositions relate to pregnancy outcomes. Here, we performed a sequential LEI in women with well-defined RIF to characterise the immune cell landscape within the endometrium through a multi-parameter flow cytometry panel that was designed to allow us to identify DC subsets; CD141 + DC and CD1c + DC (also known as DC1 and DC2, respectively), plasmacytoid DC (pDC), CD14 + macrophages, CD3 + T cells and CD56 + NK cells 32 . The aims of our study are to firstly, establish the endometrial immune milieu by interrogating composition and changes of immune cell subtypes before and after LEI in subsequent luteal phase cycles in women with RIF and secondly, to address any relationship between alterations in endometrial immune landscape and pregnancy after LEI. Results Fifty-three women with RIF were recruited of which 16 women were excluded due to poor quality samples or refusal of the repeat endometrial biopsy. Thirty seven women (age 34.6 ± 3.3 years old) underwent two sequential mid-luteal phases endometrial Pipelle biopsies prior to embryo-transfer (ET) (Fig. 1 ). Eighty-one percent of these females were ovulatory and 65% of these women had primary infertility of which majority (57%) have unknown reasons ie not attributable to various reasons as indicated in Table 2 . Thirty-three women (88%) had 2–3 IVF cycles, and the remaining 4 women had ≥4 IVF cycles with an average of 4.9 ± 1.7 previous embryo transfers (Table 1 ). Seventeen women (17/37, 46%) had successful implantation indicated by an elevated βhCG result of > 25 mIU per milliliter post IVF after the second LEI. Among these 17 women, there were 1 biochemical pregnancy, 3 miscarriages and 13 live births (35.1%). Table 1 Patient Clinical Characteristics Mean (SD) Age, years 34.6 (3.3) BMI, kg/m2 23.6 (4.4) Basal AMH (ng/mL) 4.9 (3.3) Ethnicity (n) (%) Chinese 29 (78) Malay 6 (16) Indian 1 (3) Others 1 (3) Type of infertility (n) (%) Primary 24 (65) Secondary 13 (35) Reasons for infertility (n) (%) Tubal 2 (5) Ovarian 3 (9) Endometriosis 5 (14) Uterine 2 (5) Male 4 (10) Unexplained 21 (57) Ovulatory status (n) (%) Ovulatory 30 (81) Anovulatory 7 (19) Number of previous IVF cycles (n) (%) 2 19 (51) 3 14 (38) 4 1 (3) > 5 3 (8) (value) (SD) Endometrial thickness at transfer (mm) 8.4 (0.9) Number of Embryo transfers (ET) previously 4.9 (1.7) Table 2 Antibodies used for flow cytometric analyses. Clone Source Identifier I Anti-human HLA-DR (Q.dot 605) Tu36 Invitrogen Q10052 I Anti-human CD3 FITC UCHT1 BioLegend 300406 II Anti-human CD3 PerCP/Cyanine5.5 UCHT1 BioLegend 300430 I, II Anti-human CD19 FITC HIB-19 BioLegend 302205 I Anti-human CD20 FITC 2H7 BioLegend 375508 II Anti-human CD56 (PE) W22097A BioLegend 343758 I Anti-human CD14 PE-Texas Red TuK4 Invitrogen MHCD1417 I Anti-human CD16 APC /Cyanine7 3G8 Biolegend 302018 I Anti-human CD1c PE/Cyanine7 L161 Biolegend 331516 I Anti-human CD11c Pacific Blue Bu15 Biolegend 301626 I Anti-human CD123 PerCP/Cyanine5.5 6H6 Biolegend 306016 I, II Anti-human CD45 Pacific Orange H130 Invitrogen MHCD4530 I Anti-human CD141 APC AD5-14H12 Miltenyi Biotec 130-113-314 Anti-human CD163 PE GHI/61 Biolegend 333606 Anti-human CD64 PE 10.1 Biolegend 305008 Anti-human SIRPα/β PE SE5A5 Biolegend 323806 Anti-human CD26 PE BA5b Biolegend 302706 Anti-human CD80 PE 2D10 Biolegend 305208 Anti-human CD83 PE HB15e Biolegend 305308 Anti-human CD86 PE BU63 Biolegend 374206 I used in 1st gating strategy to identify macrophages, pDCs, DCs. II used in 2nd gating to identify T, B, NK cells. Immunophenotypic assessment providing insights into the populations of DCs, macrophages, NK, B and T cells in the endometrium of the RIF population by flow cytometry (Fig. 2 ) was analysed before(1st LEI) and after the LEI (2nd LEI) procedure (Fig. 3 ). In the biopsies obtained, T-cells were the predominant immune cell (34.6 ± 19.9%@1st LEI and 40.2 ± 22.2@2nd LEI), followed by NK cells (26.2 ± 17.5% at 1st LEI and 29.1 ± 18.0% at 2nd LEI), CD14 + macrophages (7.5 ± 8.1% at 1st LEI and 7.2 ± 8.5% at 2nd LEI), B cells (2.4 ± 1.7% at 1st LEI and 3.6 ± 2.2% at 2nd LEI, CD141 + DCs (1.3 ± 1.0% at 1st LEI and 1.0 ± 0.9% at 2nd LEI and CD1c + DCs (0.8 ± 0.7% at 1st LEI and 0.6 ± 0.6% at 2nd LEI (Fig. 3 A). No significant differences were observed in the various cell types in all the paired samples we obtained (p-value:0.26 (T-cells), 0.40 (NK cells), 0.84 (CD14 + macrophages), 0.25 (B cells), 0.07 (CD141 + DCs) and 0.36 (CD1c + DCs) (Fig. 3 B-G). We did see a consistent upward trend with the lymphocytes (T, NK and B cells) and a consistent downward trend with the APCs between the 1st and 2nd LEI biopsies (Fig. 3 ). Segregating the samples based on eventual pregnant outcomes, pregnant women were younger than the non-pregnant group though not statistically significant (32.3 ± 8.1 vs 35.0 ± 3.7, p = 0.51). The proportion of the different immune cell types for the group of participants with and without a positive pregnancy outcome at the first LEI were all non-significant, indicating similarly comparable immune component prior to LEI. We next investigated if the LEI altered the immune cell milieu by comparing the cellular composition between the first and second LEI in the two groups (non-pregnant and pregnant). There was an increase in T cells in the non-pregnant participants (31.8 ± 21.7 to 44.0 ± 18.3, p-value:0.03) between the 2 LEI which was not observed in the pregnant participants (38.1 ± 19 vs 35.4 ± 26.2, p-value:0.76, Fig. 4 A). There were no differences between the T-cell proportions at 2nd LEI for the pregnant vs non-pregnant groups (35.4 ± 26.2 vs 44 ± 18.3, p-0.29). For NK cells, there is a slight increase in the non-pregnant participants (26.25 ± 17.75 to 29.15 ± 18.04, p-value:0.40, Fig. 4 B) and a reduction in the pregnant participants (29.02 ± 17.92 to 28.24 ± 19.4, p-value:0.89, Fig. 4 C). An opposite trend was observed in the CD14 + macrophages (7.50 ± 8.57 to 5.91 ± 4.56, p-value:0.44 in the non-pregnant group and 7.61 ± 7.77 to 8.89 ± 11.69, p-value:0.63, Fig. 4 D). A consistent increase was observed with B cells in both groups (2.06 ± 1.45 to 3.57 ± 3.24, p-value:0.46 in the non-pregnant group and 2.35 ± 2.05 to 3.65 ± 1.70 in the pregnant group, p-value:0.46, Fig. 4 E). A consistent decrease was observed with the DCs. The percentage of CD141 + DCs decreased from 1.38 ± 1.08 at first LEI to 0.96 ± 0.79 at second LEI and from 1.18 ± 0.87 to 1.12 ± 1.03 in the non-pregnant (p-value:0.08) and pregnant participants (p-value:0.69) (Fig. 4 F) respectively. Similarly, the CD1c + DCs decreased from 0.79 ± 0.74 at first LEI to 0.62 ± 0.61 at second LEI and from 0.75 ± 0.74 to 0.59 ± 0.52 in the non-pregnant (p-value:0.23) and pregnant participants (p-value:0.52)(Fig. 4 F) respectively. In summary, comparisons of pregnant and non-pregnant endometrial immune cells at the first or second biopsy did not reveal statistical differences in lymphocyte subpopulations (B, T and NK cells) and APCs (DCs, CD14 + macrophages) except in the T cells population amongst the non-pregnant participants (Fig. 4 ). Discussion LEI treatment in the context of RIF, two ET failures or more, has demonstrated some degree of benefit especially in the context of sequential double-luteal phase LEI 18 , 21 , 33 . Here, we took advantage of such a protocol to study the impact of LEI on the immune cell milieu in women with RIF. Our primary finding suggest that there is no change in the composition of T-cells, B-cells, Antigen-Presenting Cells and NK cells using a validated parameter flow panel 34 . We did however, show an increase in the T-cell population in women who did not become pregnant. The process of IVF is associated with overwhelming physical, emotional and financial burden especially for women with RIF. A survey by Lenson et al 2016 showed that 83% of clinicians would recommend endometrial scratching to their patients prior to IVF despite conflicting evidence 12 . Many studies are limited by their selection of women with differences in the number and timing of LEI prior to IVF 13 , 17 , 19 . Lenson et al 2019 showed that endometrial scratching did not result in a higher live birth rates than no intervention in all women undergoing IVF 20 . However, these women only received one endometrial scratch at a relatively broad time range of the menstrual cycle that is between day 3 of the preceding cycle to day 3 of the IVF cycle month itself. Further subgroup analysis did not show any benefit in the number of participants who failed implantation at least twice although this was not planned a-priori 20 . Twenty-four percent of the participants undergoing a single endometrial scratch comprising 85 and 81 participants had two or three previous unsuccessful embryo transfers respectively. Hence, the small numbers may be underpowered to statistically reflect a fifteen percent difference in live birth rates. In our study, seventeen women (46%) had a clinical pregnancy after performing two sequential endometrial scratches prior to IVF. This is consistent with a recent meta-analysis by Vitagliano et al 2018 which suggested that two luteal phase LEI is the number of interventions that gave a beneficial outcome in women with RIF undergoing IVF 18 . A meta-analysis involving 2,537 participants showed an improvement in clinical pregnancy rates for participants with 2 or more failed IVF/ ICSI cycles back in 2019 19 and an extension of it by Hoogenhuijze et al, showed in an individual participant data meta-analysis representing 4,112 participants an improved odds ratio of 1.29 of LBR 35 . Currently, there is no consensus for the definition of RIF due to the clinical heterogenicity of studies performed. Our selection criteria is consistent with a recent systemic review by Polanski et al which defined RIF as the absence of implantation after two consecutive cycles of IVF where the cumulative number of transferred embryos was no less than four for cleavage-stage embryos 9 . A uniform and clear definition of RIF is important as it will aid the counselling and expectation of couples undergoing repeated IVF cycles to achieve a successful pregnancy. In addition, it will clearly define the parameters for which LEI improves pregnancy outcomes as what we have observed in this cohort and our previous study 23 . The immune landscape in the endometrium is dynamic with an influx of macrophages, DCs and NK cells during the secretory phase of the menstrual cycle 36 . The low number of CD45 + cells in the endometrium in the early follicular to early secretory phase undergoes a five-fold increase during the secretory phase 37 , peaking at the late secretory phase 38 . Within the innate immune system, uterine NK (uNK) cells and APCs including macrophages and DCs comprise a large proportion of the decidual leukocyte population and they are known to play important roles in modulating trophoblast invasion, inflammation, angiogenesis and vascular remodelling during implantation 39 , 40 . Patients with RIF have been shown to have significantly raised NK and B cells 41 , 42 , suggesting the presence of a potential differential pro-inflammatory bias in RIF patients. Unravelling the mechanism behind LEI will also be beneficial in explaining the observable benefits of LEI and in this study, we looked into the immune milieu of the endometrial biopsies for answers. LEI induces an inflammatory response that promotes implantation although its exact mechanism is not elucidated 43 . In our study, we showed that T-cells were the predominant immune cell, followed by NK cells and CD14 + macrophages in the endometrium of the cohort of RIF patients. This is congruent with Givan et al where it was established back in 2011 that CD3 + T cells constitutes approximately 40% of the CD45 + cells in the uterine endometrium in both proliferative and secretory phase and estimated the proportion of NK cells to be around 25% of the CD45 + leukocytes 44 . Flynn et al however, found that T cells population vary throughout the menstrual cycle, comprise 40–60% of the total leukocyte population in the proliferative phase and drops to < 10% in the late luteal phase in healthy women 37 . T cells are activated by the innate immune system and are effector cells of the adaptive immune system that affect the activity of other immune cells through secreted cytokines that mount an immune response against pathogens 45 . Our study showed instead that T cells were the predominant immune cell population in the luteal phase in our population of women with RIF. Interestingly, within the non-pregnant population, there is a further and significant increase in the T cell population after the LEI, possibly indicating further increased inflammation in the endometrium. On the contrary, that was not observed in the pregnant group. This observation is congruent with Ganeva and colleagues’ findings where there were significantly lower CD3 + T cells in RIF women with a successful implantation than those who were unsuccessful 46 . However, comparison of the T cell population in the 2nd LEI sample between the pregnant and non-pregnant group in our study did not reveal any differences. This could be due to the large inter-patient variability, with a small sample size of 20 in the non-pregnant and 17 in the pregnant group. Nonetheless, our data along with others 46 suggest that the LEI could have set off another mechanism to inhibit the T cell from further increase in women who respond positively. Decidual NK cells contribute to pregnancy by increasing the blood flow through remodeling of maternal arterioles at the feto-maternal interface and helping the migration of the trophoblast. NK cells is also responsible for the secretion of angiogenic factors such as vascular endothelial growth factor and angiopoietin-2, as well as cytokines and growth factors such as TNF-α, IL-10, GM-CSF, placental growth factor, IL-1β, TGF-β1, CSF-1, LIF, and IFN-γ 47 . Therefore, their distribution and activation at the time of fertilization and implantation plays a critical role in pregnancy outcome 48 . NK cells account for 20% of lymphocytes in the proliferative phase and increases to 40–50% in the luteal phase and a maximum of 70–80% during decidualization 49 , 50 . In contrast, NK cells represent approximately 26–29% of CD45 + leukocytes in our RIF population, as opposed to the nearly 70% of endometrial leucocytes in the late luteal phase or early pregnancy 51 , 52 . This corroborated with other studies on women with RIF 53 – 55 and corresponds well with the postulation that women with subfertility may have lower uNK cells compared to healthy women. From our results, LEI does not alter the proportion of NK cells in women with RIF. NK cell subpopulations are identified by differential expression of a range of NK receptors 56 whereby circulating NK cells are primarily CD56 dim CD16 bright whereas uNK cells are mostly CD56 bright CD16 dim 57 . However, we did not distinguish this NK subpopulation which is a limitation of our study. The next most abundant cell types are B cells, which are known to be found throughout the cycle, at low levels, corroborating with our findings of approximately 2% of the CD45 + cell population 38 , 58 . With their low levels, they have long been perceived as insignificant until more recently where they are shown to have distinct characteristics and may be actively involved in shaping the endometrial immune environment 59 . The quantity of B cells within the endometrium is altered in certain pathologies in presence of endometrial inflammation 59 . There are conflicting findings as to whether RIF patients have increased B-cell numbers 59 . In our study, within our definition of RIF patients, we showed an increase of 1.2% (50%) increase in the proportion of B cells within the entire cohort though non statistically significant. We observed a similar increase with B cells in both the non-pregnant and pregnant cohort (Fig. 4 E), suggesting an inflammatory response with the LEI. A wide patient heterogeneity as well as a small sample size could have attributed to the statistical significance and more should be done to look at this. Dendritic cells are APCs that process and present antigenic materials to T lymphocytes 60 , 61 and bridge the innate and adaptive immune systems 62 , 63 . We noted a trend towards a reduction of both CD141 + DCs and CD1c + DCs though non statistically significant. Both CD141 + and CD1c + DCs have a common origin of myeloid precursors and produces pro-inflammatory IL-1B, 6, 8 and TNF 64 . They are also responsible for taking up, processing and presenting protein antigens to CD4 + and CD8 + T cells 64 . During pregnancy, the maternal-fetal interface undergoes dynamic changes to allow the fetus to grow and develop in the uterus, despite being recognized by the maternal immune cells. Our observation of the reduction in both DCs subtypes following an LEI may not have an impact difference as it is statistically non-significant but if that is due to the power of the study, this reduction could mean aiding in immune tolerance toward the semi-allogeneic fetus, contributing to the beneficial effects of LEI for women with RIF. Analysis of rare cells in the endometrium has its own set of challenges of weighing between the scientific benefits provided by a large sample volume versus the clinical and ethical difficulties of obtaining tissue 65 . A larger sample size, or the use of newer high resolution technologies like single cell analyses may be required to illuminate this field. Overall, we did not find any significant changes of immune cell subtypes before and after LEI in subsequent luteal phase cycles in women with RIF. No significant differences were observed in DCs, macrophages, NK cells and B cells between the first and second Pipelle biopsy which is congruent with Ganeva at el where there were non-significant differences in median of percentages of T cells, NK cells, macrophages, monocytes and B cells 46 . Of particular interest, both their and our study showed a significant change in the T cells, although our participants of interest are different, theirs being women with RIF with and without a successful implantation while ours are women with RIF undergoing LEI without and without getting pregnant 46 . The immune landscape is different in women with RIF as compared to normal women. Endometrial immune dysregulation is also implicated in women with different pathologies including recurrent pregnancy loss, preterm labour and endometriosis 66 – 68 . In our study, twenty one (57%) and five women (14%) had unexplained fertility and endometriosis respectively. The peripheral and endometrial immune system are altered in women with endometriosis with macrophages, immature DCs and regulatory T cells behaving differently 68 – 70 and that can confound our results, exacerbated by the small samples size. Immune activation or imbalance between immune effectors and regulators has been reported in the patients with reproductive failure hence the importance of this study 52 . That said, the etiology of RIF is likely multifactorial and thus, the mechanism(s) for an improvement in clinical pregnancy rates brought about by LEI may not only be immunologically based or even if it is, may not be directly measured through the differential proportion of the cells types we looked at. The mechanical abrasion followed by wound healing may positively influence implantation rates by altering the release of cytokines, chemokines, IL-15, adhesion molecules, growth factors and hormones 41 or switching on and/or activating dormant genes 14 , 71 . As the human endometrium undergoes more than 400 cycles of regeneration, differentiation and shedding during a women's reproductive life span 72 , it is also feasible that menstruation may bestow plasticity on the uterus through activation of endometrial stem cells. This may be timely for tissue remodelling associated with deep placentation and enhancing embryonic implantation for pregnancy 73 . We have previously shown that the decidual response is seen at the embryo-maternal interface with differentiating perivascular stem cells establishing distinct cytokine and chemokine profiles that affect local immune responses in pregnancy 74 . We have also shown that endometrial mesenchymal stem/ progenitor cells derived from the second LEI tend to be more proliferative with a shorter doubling time, with a reduced decidualization propensity 23 . The novelty of our study includes the approach to LEI involving the same patient over consecutive menstrual cycles where the use of uterine catheter acts as intervention while concurrently allows tissue collection. Patients also act as their own control which can reduce inter-patient variability that can confound results. In addition, we employed a validated assay (Fig. 2 ) that can interrogate the composition and content of the various immune cell subtypes with high specificity and sensitivity, circumventing the limitations of traditional methods analysis 75 , 76 . The approach of treating endometrial specimens as a cell suspension is a powerful approach as this technique has significant advantages over the traditional immuno-histochemical staining methods, including the ability to precisely interrogate multiple markers simultaneously from a single specimen and exclude false positive or negative signals from non-viable cells. That said, limitations of our study include a small sample size of fifty-three women with a high drop-out rate of sixteen patients (30%) and that our results are dependent on the Pipelle biopsies obtained which are beset by different sampling regions between patients as well as between the first and second LEI. In addition, we did not perform subset analysis of the NK (uterine and non -uterine) and T cells. T cell subsets such as CD4, CD8 and T regulatory cells and their activation status which are implicated in patients with subfertility can further ascertain better understanding on the complex interactions between the innate and adaptive immune system during implantation and pregnancy. In this small but well-defined RIF population where sequential paired samples were analysed by high resolution flow cytometry, the immune landscape had remained largely unchanged post LEI in women with RIF. Further research in this area, including uncovering the immune activation statuses of NK and T-cells is needed. These can be done by transcriptomics, applying RNA sequencing for gene expression analysis in a single cell manner and/or high-resolution cytometry by time-of-flight (CYTOF). Elucidating the critical pathways surrounding the beneficial effects of LEI in RIF patients will not only offer a deeper understanding of the critical events surrounding human implantation but opens the possibility of devising approaches to enhance fertility outcomes. Materials and Methods Ethics Approval The current study was reviewed and approved by the SingHealth Centralized Institutional Review Board (CIRB 2013/215/D). Informed written consent was obtained for all participants before their participation in the study at KKIVF Centre, KK Women’s and Children’s Hospital. All experimental procedures were performed in accordance with the relevant guidelines and regulations. Inclusion and Exclusion Criteria Our inclusion criteria are women with RIF undergoing IVF with RIF defined as failure to achieve a clinical pregnancy after a transfer of minimum four good-quality embryos in a minimum of two or more previous failed embryo transfers. Other inclusion factors include women 7 mm at embryo transfer and a normal hormonal profile. Our exclusion criteria included women > 40 years old, BMI > 35 and women who do not meet our criteria for RIF or women who declined a repeat endometrial scratch in the following menstrual cycle. Serum progesterone was measured to check for ovulatory status. Serum beta human chorionic gonadotrophin (βhCG) was measured 17 days after IVF embryo transfer to determine if women became pregnant with a positive βhCG result > 25 mIU per milliliter. Clinical pregnancy rate was defined as the presence of intrauterine gestation sac on ultrasound scan 2 weeks after a positive βhCG result. Sample Collection and Processing To characterize the immunological landscape alterations in LEI, we performed sequential implantation-phase (mid-luteal) endometrial biopsies (first and second biopsies) in RIF patients prior to IVF. These women underwent two consecutive LEI with a Pipelle endometrial catheter to obtain endometrial tissue during the first and succeeding mid-luteal phase (Day 21–25) of their menstrual cycles (Fig. 1 ). The endometrial scratch procedure was performed by clinic doctors using a Pipelle, a plastic biopsy catheter approximately 3 mm in diameter (Pipelle de cornier, Laboratoire CCD, France). Endometrial tissues obtained were minced into fragments and digested for 30 min at 37°C with 1 mg/mL of collagenase type IV (Sigma-Aldrich, St Louis, MO), 0.2 mg/mL of DNase (Roche Applied Science, Germany) and 1 mg/mL of bovine serum albumin in PBS. The single cell suspension was passed through a mesh, washed in PBS, treated with Ammonium-Chloride-Potassium lysing buffer (0.01 M KHCO3 buffer, 0.16M NH4Cl and 0.1 mM ethylenediaminetetraacetic acid (EDTA) to remove red blood cells, washed and resuspended in PBS as previously described 43 . Flow Cytometry Cells were blocked in human blocking buffer (5% human serum (Sigma-Aldrich), 1% rat serum (Sigma-Aldrich), 1% mouse serum (Sigma-Aldrich), 5% FBS, and 2 mM EDTA) for 15 min at 4°C and incubated with antibody cocktails (Table 1 ) for 30 min at 4°C. Cell suspensions were stained for viability with 1:3,000 DAPI (Sigma-Aldrich). Flow cytometric analyses were performed on an LSR II (Becton Dickinson) and data analysed with FlowJo software (TreeStar). Two antibody cocktails were used. Live cells (DAPI − ) were first selected. Doublets were then removed and leukocytes (CD45 + ) identified. With the first antibody cocktail, tissue CD14 + macrophages, pDCs, CD141 + DCs, CD1c + DCs were identified (Fig. 2Ai). Antigen presenting cells were identified as HLA-DR + and lineage (CD3, CD19, CD20) − . CD14 + macrophages which also express the macrophage markers CD163 and CD64 were enumerated. From the CD14-CD16- fraction, pDCs were identified by CD123 + and CD11c − . From the CD123 − CD11c − population, while CD141 + CD1c − and CD1c + CD141 − were enumerated. With the second antibody cocktail, CD3 + T cells, CD19 + CD20 + B cells and CD56 + NK cells were identified and enumerated (Fig. 2Aii). Statistical Analysis The data is presented as mean ± standard deviation. Statistical analysis comparing first and second LEI was performed using non-parametric (Mann-Whitney for unpaired or Wilcoxon signed-ranked test for paired data) or Student’s t-test where appropriate with Graphpad Prism 10. A two-sided p-value at the 5% level was considered to indicate statistical significance. Declarations Funding Declarations This study was supported by the KK Women’s and Children’s Hospital Health Endowment Fund (KKHHEF/2013/07) and National Medical Research council (NMRC) seed fund (0004/2017). JKYC is supported by National Medical Research Council (CIRG/1484/2018, NMRC CSA (SI)/008/2016, CIRG21jun-0045 and STaR22jul-0004). Additional Information The authors declare no conflicts of interest. Author Contribution RWKL, NM, FG, YHL and JKYC study conceptualization. AM and NM designed and performed the flow experiments. RWKL, YF, YHL analysed the data and drafted the paper. RWKL, TYT, JKYC obtained funding, consented patients and collected data. All authors read, reviewed and approved the final version of the article. Acknowledgement The authors wish to thank Dr Sadhana Nadarajah, Dr Tan Heng Hao, Miss Ng Xiang Wen from KK Women’s and Children’s Hospital for clinical administrative support, Mr Gurmit Singh from Singapore Immunology Network (SIgN) for tissue sample processing and flow cytometry analysis. Data Availability The authors confirm that the data supporting the findings are available within the article. Please contact the corresponding author for more information. References Luke, B. et al. Cumulative birth rates with linked assisted reproductive technology cycles. N Engl. J. Med. 366 , 2483–2491. 10.1056/NEJMoa1110238 (2012). Karimzadeh, M. A., Ayazi Rozbahani, M. & Tabibnejad, N. 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. 10.1111/j.1479-828X.2009.01076.x (2009). Revel, A. 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19:21:00","extension":"png","order_by":11,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":56256,"visible":true,"origin":"","legend":"","description":"","filename":"OnlineFig1.png","url":"https://assets-eu.researchsquare.com/files/rs-7494176/v1/93d27e129a443d7e31e98bff.png"},{"id":93715068,"identity":"c50f276e-a7ae-447d-941b-a04832a91e44","added_by":"auto","created_at":"2025-10-16 19:21:01","extension":"png","order_by":12,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":539019,"visible":true,"origin":"","legend":"","description":"","filename":"OnlineFig2.png","url":"https://assets-eu.researchsquare.com/files/rs-7494176/v1/0920e11b6f76c6ef76c51a3a.png"},{"id":93715053,"identity":"688eefbd-d934-47c4-97a1-00bc4895b231","added_by":"auto","created_at":"2025-10-16 19:21:00","extension":"png","order_by":13,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":115636,"visible":true,"origin":"","legend":"","description":"","filename":"OnlineFig3.png","url":"https://assets-eu.researchsquare.com/files/rs-7494176/v1/30fa4dbcadaec22f8e2dc8fb.png"},{"id":93715061,"identity":"76fba7dd-847d-4d55-bd7d-3ee305108b10","added_by":"auto","created_at":"2025-10-16 19:21:00","extension":"png","order_by":14,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":378889,"visible":true,"origin":"","legend":"","description":"","filename":"OnlineFig4.png","url":"https://assets-eu.researchsquare.com/files/rs-7494176/v1/61dd0a8a2e044630e15e3c31.png"},{"id":93715455,"identity":"605a3032-5c84-4496-abcd-35c313764834","added_by":"auto","created_at":"2025-10-16 19:29:00","extension":"xml","order_by":15,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":174402,"visible":true,"origin":"","legend":"","description":"","filename":"fbc637d826ad4a79a2ce5411b516eb8f1structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-7494176/v1/77a4bf38d78a77d181f46715.xml"},{"id":93715052,"identity":"ca057403-459a-4da2-a112-8d0faa077ccd","added_by":"auto","created_at":"2025-10-16 19:21:00","extension":"html","order_by":16,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":191863,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7494176/v1/aed03c5ff77539cd012d1465.html"},{"id":93715043,"identity":"2c9d0f98-5359-4fb3-b2e4-6d82a696a0be","added_by":"auto","created_at":"2025-10-16 19:20:59","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":284021,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eConsecutive Pipelle biopsies performed during mid luteal phases prior to IVF.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Fig1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7494176/v1/c543d87fb3a23a0a03d9b2f5.jpg"},{"id":93715051,"identity":"21b0a335-b279-43a5-9fe4-c85f36a2b461","added_by":"auto","created_at":"2025-10-16 19:21:00","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1222544,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFlow cytometric gating strategy used to identify mononuclear phagocyte and lymphocyte populations. \u0026nbsp;\u003c/strong\u003eLive cells (DAPI\u003csup\u003e-\u003c/sup\u003e) were first selected. Doublets were removed and leukocytes (CD45\u003csup\u003e+\u003c/sup\u003e) were identified (A). Mononuclear phagocytes were identified as HLA-DR\u003csup\u003e+ \u003c/sup\u003eand lineage (CD3, CD19, CD20)\u003csup\u003e-\u003c/sup\u003e. pDC and DC are CD14\u003csup\u003e-\u003c/sup\u003e and CD16\u003csup\u003e-\u003c/sup\u003e. pDC are CD123\u003csup\u003e+\u003c/sup\u003e and CD11c\u003csup\u003e-\u003c/sup\u003e. CD141\u003csup\u003e+\u003c/sup\u003eDC are CD1c\u003csup\u003e-\u003c/sup\u003e. CD1c\u003csup\u003e+\u003c/sup\u003e DC are CD141\u003csup\u003e-\u003c/sup\u003e. T cells express CD3. B cells express CD19 and CD20. NK cells express CD56. Representative histograms used to further characterise the cellular populations, from n=1-3 experiments (B). CD14\u003csup\u003e+\u003c/sup\u003e macrophages also express CD163, CD64, SIRPa/b and CD86(Fig 2B). Fluorescence minus one (FMO) – negative control.\u003c/p\u003e","description":"","filename":"Fig2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7494176/v1/f60f632de94c1caf81d96076.jpg"},{"id":93715049,"identity":"a5d322bd-b9da-4eaa-b743-c0b97e05fd56","added_by":"auto","created_at":"2025-10-16 19:21:00","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":558212,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eImmune cell populations in the endometrial biopsy. \u003c/strong\u003eBreakdown of the immune cell types in the biopsies (A). Comparisons of paired samples for T cells (B), NK cells (C), CD14+ macrophages (D), B cells (E), CD141+ DCs (F) and CD1c+ DCs (G).\u003c/p\u003e","description":"","filename":"Fig3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7494176/v1/5f82ec7b5df2d46f34813518.jpg"},{"id":93715042,"identity":"8dfe9abf-fee7-414b-9642-cd3dea7cc1ff","added_by":"auto","created_at":"2025-10-16 19:20:59","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":541406,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDistributions of immune cells. \u003c/strong\u003eT-cells (A), NK (B), macrophages (C), B-Cells (D), CD141+ DCs (E) and CD1c+ DCs (F) segregated by pregnant and non-pregnant cohorts.\u003c/p\u003e","description":"","filename":"Fig4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7494176/v1/189a75aa8308479409d9aa77.jpg"},{"id":99545340,"identity":"22525c4f-30fd-4df7-82d0-717471c5c81b","added_by":"auto","created_at":"2026-01-05 16:06:04","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3649294,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7494176/v1/6fdc1e53-34e5-477e-83cf-c749823239a9.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"The Immune Milieu After Local Endometrial Injury in Women with Recurrent Implantation Failure","fulltext":[{"header":"Introduction","content":"\u003cp\u003eAdvancements in reproductive techniques have improved the live birth rates of in-vitro fertilization (IVF) cycles. However, live-birth rates of IVF have plateaued around 30%\u003csup\u003e1\u003c/sup\u003e for over a decade despite extensive research into embryonic factors and endometrial receptivity\u003csup\u003e\u003cspan additionalcitationids=\"CR3\" citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. Embryo implantation\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e remains the rate limiting step in IVF especially in a small group of women with recurrent implantation failure (RIF). The definition of RIF is currently not universally defined. Proposed RIF definitions include failure to achieve a clinical pregnancy after transferring at least four good quality embryos following two to six embryo-transfers (ET)\u003csup\u003e\u003cspan additionalcitationids=\"CR7 CR8\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eIt was previously observed that local endometrial injury (LEI) by endometrial scratching before IVF led to a two-fold increase in pregnancy rates\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e,\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. Endometrial scratch involves obtaining tissue biopsy using a sampler such as a Pipelle catheter where a \u0026lsquo;scratch\u0026rsquo; is performed that is reasonably well tolerated without any need for analgesia\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. Since then, there have been many randomized clinical trials evaluating the impact of LEI on pregnancy rates prior to IVF\u003csup\u003e\u003cspan additionalcitationids=\"CR14 CR15\" citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. In the presence of inconclusive and contrasting evidence, there is no current consensus whether patients should receive endometrial scratching prior to IVF\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. The majority of randomized controlled trials (RCTs) showed a general trend towards benefit of LEI especially where the number of previous failures are more than two. In order to improve the power to detect possible differences, meta-analyses conducted by both Vitagliano et al and van Hoogenhuijze et al both demonstrated improved live-birth rates post LEI in women with multiple failures\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e,\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. One RCT that stood out was the PIP trial that did not show a benefit in a subgroup of women with two or more failures\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. However, the PIP trial was designed to test the effect of LEI in a unselected group of patients and not women with RIF. The most recent large RCT, SCRaTCH trial, showed a trend towards benefit after one IVF cycle failure and with a single luteal phase LEI\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. A more updated meta-analyses provided by Vitagliano et al that included both the PIP and SCRaTCH trial continued to show a benefit of Live Births (RR 1.21, 95CI 1.05\u0026ndash;1.40)\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. The timing, frequency and type of LEI instituted varied considerably between the various studies, making it difficult to generalize the findings\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e,\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e,\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. It is therefore important and timely to define the type of patients being offered this intervention, the way LEI is administered in this specific group of patients with RIF to maximize their chances of getting pregnant.\u003c/p\u003e\u003cp\u003ePutative mechanisms on how LEI may improve implantation rates in patients with RIF are currently unknown. Some proposed mechanisms include increasing endometrial receptivity by altering decidualization favouring implantation\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e, provoking the endometrial immune system to generate an inflammatory response\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e leading to increased secreted cytokines, growth factors and upregulation of adhesion molecules \u003csup\u003e\u003cspan additionalcitationids=\"CR25\" citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. The feto-maternal immune cross talk is complex with recruitment and modification of endometrial immune cells involved in implantation\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. Research into the immune milieu in the endometrium have shown that the roles of decidual immune cells are associated closely with implantation, angiogenesis and maintenance of pregnancy\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e,\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. These include macrophages, dendritic cells (DCs), natural killer (NK) cells, lymphocytes such as B cells and T cells which are involved with implantation and feto-maternal tolerance towards pregnancy\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e,\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eWe have previously studied the effect of LEI on SUSD2\u0026thinsp;+\u0026thinsp;mesenchymal stromal cells using a sequential mid-luteal Pipelle approach\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. However, the effects on the various subsets of immune and APCs in subsequent menstrual cycles are unknown. To date, there has been no properly performed study that characterizes tissue resident immune cell sub-sets before and after LEI in subsequent luteal phase in women with RIF, and how any alterations in immune cell types and compositions relate to pregnancy outcomes. Here, we performed a sequential LEI in women with well-defined RIF to characterise the immune cell landscape within the endometrium through a multi-parameter flow cytometry panel that was designed to allow us to identify DC subsets; CD141\u0026thinsp;+\u0026thinsp;DC and CD1c\u0026thinsp;+\u0026thinsp;DC (also known as DC1 and DC2, respectively), plasmacytoid DC (pDC), CD14\u0026thinsp;+\u0026thinsp;macrophages, CD3\u0026thinsp;+\u0026thinsp;T cells and CD56\u0026thinsp;+\u0026thinsp;NK cells\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. The aims of our study are to firstly, establish the endometrial immune milieu by interrogating composition and changes of immune cell subtypes before and after LEI in subsequent luteal phase cycles in women with RIF and secondly, to address any relationship between alterations in endometrial immune landscape and pregnancy after LEI.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eFifty-three women with RIF were recruited of which 16 women were excluded due to poor quality samples or refusal of the repeat endometrial biopsy. Thirty seven women (age 34.6\u0026thinsp;\u0026plusmn;\u0026thinsp;3.3 years old) underwent two sequential mid-luteal phases endometrial Pipelle biopsies prior to embryo-transfer (ET) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Eighty-one percent of these females were ovulatory and 65% of these women had primary infertility of which majority (57%) have unknown reasons ie not attributable to various reasons as indicated in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. Thirty-three women (88%) had 2\u0026ndash;3 IVF cycles, and the remaining 4 women had \u0026ge;4 IVF cycles with an average of 4.9\u0026thinsp;\u0026plusmn;\u0026thinsp;1.7 previous embryo transfers (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Seventeen women (17/37, 46%) had successful implantation indicated by an elevated βhCG result of \u0026gt;\u0026thinsp;25 mIU per milliliter post IVF after the second LEI. Among these 17 women, there were 1 biochemical pregnancy, 3 miscarriages and 13 live births (35.1%).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003ePatient Clinical Characteristics\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"2\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eMean (SD)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eAge, years\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e34.6 (3.3)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eBMI, kg/m2\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e23.6 (4.4)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eBasal AMH (ng/mL)\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e4.9 (3.3)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eEthnicity\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003e(n) (%)\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eChinese\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e29 (78)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMalay\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e6 (16)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eIndian\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1 (3)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eOthers\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1 (3)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eType of infertility\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003e(n) (%)\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePrimary\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e24 (65)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSecondary\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e13 (35)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eReasons for infertility\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003e(n) (%)\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTubal\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e2 (5)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eOvarian\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e3 (9)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eEndometriosis\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e5 (14)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eUterine\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e2 (5)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMale\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e4 (10)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eUnexplained\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e21 (57)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eOvulatory status\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003e(n) (%)\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eOvulatory\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e30 (81)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAnovulatory\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e7 (19)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eNumber of previous IVF cycles\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003e(n) (%)\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e19 (51)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e14 (38)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1 (3)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u0026gt;\u0026thinsp;5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e3 (8)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003e(value) (SD)\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eEndometrial thickness at transfer (mm)\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e8.4 (0.9)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eNumber of Embryo transfers (ET) previously\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e4.9 (1.7)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eAntibodies used for flow cytometric analyses.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"4\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eClone\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eSource\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eIdentifier\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003csup\u003eI\u003c/sup\u003e Anti-human HLA-DR (Q.dot 605)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eTu36\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eInvitrogen\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eQ10052\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003csup\u003eI\u003c/sup\u003e Anti-human CD3 FITC\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eUCHT1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eBioLegend\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e300406\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003csup\u003eII\u003c/sup\u003eAnti-human CD3 PerCP/Cyanine5.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eUCHT1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eBioLegend\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e300430\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003csup\u003eI, II\u003c/sup\u003eAnti-human CD19 FITC\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eHIB-19\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eBioLegend\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e302205\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003csup\u003eI\u003c/sup\u003e Anti-human CD20 FITC\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e2H7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eBioLegend\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e375508\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003csup\u003eII\u003c/sup\u003e Anti-human CD56 (PE)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eW22097A\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eBioLegend\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e343758\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003csup\u003eI\u003c/sup\u003e Anti-human CD14 PE-Texas Red\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eTuK4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eInvitrogen\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eMHCD1417\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003csup\u003eI\u003c/sup\u003e Anti-human CD16 APC /Cyanine7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e3G8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eBiolegend\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e302018\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003csup\u003eI\u003c/sup\u003e Anti-human CD1c PE/Cyanine7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eL161\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eBiolegend\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e331516\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003csup\u003eI\u003c/sup\u003e Anti-human CD11c Pacific Blue\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eBu15\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eBiolegend\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e301626\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003csup\u003eI\u003c/sup\u003e Anti-human CD123 PerCP/Cyanine5.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e6H6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eBiolegend\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e306016\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003csup\u003eI, II\u003c/sup\u003eAnti-human CD45 Pacific Orange\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eH130\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eInvitrogen\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eMHCD4530\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003csup\u003eI\u003c/sup\u003e Anti-human CD141 APC\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eAD5-14H12\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eMiltenyi Biotec\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e130-113-314\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAnti-human CD163 PE\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eGHI/61\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eBiolegend\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e333606\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAnti-human CD64 PE\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e10.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eBiolegend\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e305008\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAnti-human SIRPα/β PE\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eSE5A5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eBiolegend\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e323806\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAnti-human CD26 PE\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eBA5b\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eBiolegend\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e302706\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAnti-human CD80 PE\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e2D10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eBiolegend\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e305208\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAnti-human CD83 PE\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eHB15e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eBiolegend\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e305308\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAnti-human CD86 PE\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eBU63\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eBiolegend\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e374206\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"4\"\u003e\u003csup\u003e\u003cem\u003eI\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eused in 1st gating strategy to identify macrophages, pDCs, DCs.\u003c/em\u003e \u003csup\u003e\u003cem\u003eII\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eused in 2nd gating to identify T, B, NK cells.\u003c/em\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eImmunophenotypic assessment providing insights into the populations of DCs, macrophages, NK, B and T cells in the endometrium of the RIF population by flow cytometry (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e was analysed before(1st LEI) and after the LEI (2nd LEI) procedure (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). In the biopsies obtained, T-cells were the predominant immune cell (34.6\u0026thinsp;\u0026plusmn;\u0026thinsp;19.9%@1st LEI and 40.2\u0026thinsp;\u0026plusmn;\u0026thinsp;22.2@2nd LEI), followed by NK cells (26.2\u0026thinsp;\u0026plusmn;\u0026thinsp;17.5% at 1st LEI and 29.1\u0026thinsp;\u0026plusmn;\u0026thinsp;18.0% at 2nd LEI), CD14\u0026thinsp;+\u0026thinsp;macrophages (7.5\u0026thinsp;\u0026plusmn;\u0026thinsp;8.1% at 1st LEI and 7.2\u0026thinsp;\u0026plusmn;\u0026thinsp;8.5% at 2nd LEI), B cells (2.4\u0026thinsp;\u0026plusmn;\u0026thinsp;1.7% at 1st LEI and 3.6\u0026thinsp;\u0026plusmn;\u0026thinsp;2.2% at 2nd LEI, CD141\u0026thinsp;+\u0026thinsp;DCs (1.3\u0026thinsp;\u0026plusmn;\u0026thinsp;1.0% at 1st LEI and 1.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.9% at 2nd LEI and CD1c\u0026thinsp;+\u0026thinsp;DCs (0.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.7% at 1st LEI and 0.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.6% at 2nd LEI (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). No significant differences were observed in the various cell types in all the paired samples we obtained (p-value:0.26 (T-cells), 0.40 (NK cells), 0.84 (CD14\u0026thinsp;+\u0026thinsp;macrophages), 0.25 (B cells), 0.07 (CD141\u0026thinsp;+\u0026thinsp;DCs) and 0.36 (CD1c\u0026thinsp;+\u0026thinsp;DCs) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB-G). We did see a consistent upward trend with the lymphocytes (T, NK and B cells) and a consistent downward trend with the APCs between the 1st and 2nd LEI biopsies (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eSegregating the samples based on eventual pregnant outcomes, pregnant women were younger than the non-pregnant group though not statistically significant (32.3\u0026thinsp;\u0026plusmn;\u0026thinsp;8.1 vs 35.0\u0026thinsp;\u0026plusmn;\u0026thinsp;3.7, p\u0026thinsp;=\u0026thinsp;0.51). The proportion of the different immune cell types for the group of participants with and without a positive pregnancy outcome at the first LEI were all non-significant, indicating similarly comparable immune component prior to LEI. We next investigated if the LEI altered the immune cell milieu by comparing the cellular composition between the first and second LEI in the two groups (non-pregnant and pregnant). There was an increase in T cells in the non-pregnant participants (31.8\u0026thinsp;\u0026plusmn;\u0026thinsp;21.7 to 44.0\u0026thinsp;\u0026plusmn;\u0026thinsp;18.3, p-value:0.03) between the 2 LEI which was not observed in the pregnant participants (38.1\u0026thinsp;\u0026plusmn;\u0026thinsp;19 vs 35.4\u0026thinsp;\u0026plusmn;\u0026thinsp;26.2, p-value:0.76, Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). There were no differences between the T-cell proportions at 2nd LEI for the pregnant vs non-pregnant groups (35.4\u0026thinsp;\u0026plusmn;\u0026thinsp;26.2 vs 44\u0026thinsp;\u0026plusmn;\u0026thinsp;18.3, p-0.29). For NK cells, there is a slight increase in the non-pregnant participants (26.25\u0026thinsp;\u0026plusmn;\u0026thinsp;17.75 to 29.15\u0026thinsp;\u0026plusmn;\u0026thinsp;18.04, p-value:0.40, Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB) and a reduction in the pregnant participants (29.02\u0026thinsp;\u0026plusmn;\u0026thinsp;17.92 to 28.24\u0026thinsp;\u0026plusmn;\u0026thinsp;19.4, p-value:0.89, Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). An opposite trend was observed in the CD14\u0026thinsp;+\u0026thinsp;macrophages (7.50\u0026thinsp;\u0026plusmn;\u0026thinsp;8.57 to 5.91\u0026thinsp;\u0026plusmn;\u0026thinsp;4.56, p-value:0.44 in the non-pregnant group and 7.61\u0026thinsp;\u0026plusmn;\u0026thinsp;7.77 to 8.89\u0026thinsp;\u0026plusmn;\u0026thinsp;11.69, p-value:0.63, Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). A consistent increase was observed with B cells in both groups (2.06\u0026thinsp;\u0026plusmn;\u0026thinsp;1.45 to 3.57\u0026thinsp;\u0026plusmn;\u0026thinsp;3.24, p-value:0.46 in the non-pregnant group and 2.35\u0026thinsp;\u0026plusmn;\u0026thinsp;2.05 to 3.65\u0026thinsp;\u0026plusmn;\u0026thinsp;1.70 in the pregnant group, p-value:0.46, Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE). A consistent decrease was observed with the DCs. The percentage of CD141\u0026thinsp;+\u0026thinsp;DCs decreased from 1.38\u0026thinsp;\u0026plusmn;\u0026thinsp;1.08 at first LEI to 0.96\u0026thinsp;\u0026plusmn;\u0026thinsp;0.79 at second LEI and from 1.18\u0026thinsp;\u0026plusmn;\u0026thinsp;0.87 to 1.12\u0026thinsp;\u0026plusmn;\u0026thinsp;1.03 in the non-pregnant (p-value:0.08) and pregnant participants (p-value:0.69) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF) respectively. Similarly, the CD1c\u0026thinsp;+\u0026thinsp;DCs decreased from 0.79\u0026thinsp;\u0026plusmn;\u0026thinsp;0.74 at first LEI to 0.62\u0026thinsp;\u0026plusmn;\u0026thinsp;0.61 at second LEI and from 0.75\u0026thinsp;\u0026plusmn;\u0026thinsp;0.74 to 0.59\u0026thinsp;\u0026plusmn;\u0026thinsp;0.52 in the non-pregnant (p-value:0.23) and pregnant participants (p-value:0.52)(Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF) respectively. In summary, comparisons of pregnant and non-pregnant endometrial immune cells at the first or second biopsy did not reveal statistical differences in lymphocyte subpopulations (B, T and NK cells) and APCs (DCs, CD14\u003csup\u003e+\u003c/sup\u003e macrophages) except in the T cells population amongst the non-pregnant participants (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eLEI treatment in the context of RIF, two ET failures or more, has demonstrated some degree of benefit especially in the context of sequential double-luteal phase LEI\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e,\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e,\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. Here, we took advantage of such a protocol to study the impact of LEI on the immune cell milieu in women with RIF. Our primary finding suggest that there is no change in the composition of T-cells, B-cells, Antigen-Presenting Cells and NK cells using a validated parameter flow panel\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. We did however, show an increase in the T-cell population in women who did not become pregnant.\u003c/p\u003e\u003cp\u003eThe process of IVF is associated with overwhelming physical, emotional and financial burden especially for women with RIF. A survey by Lenson et al 2016 showed that 83% of clinicians would recommend endometrial scratching to their patients prior to IVF despite conflicting evidence\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. Many studies are limited by their selection of women with differences in the number and timing of LEI prior to IVF\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e,\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e,\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. Lenson et al 2019 showed that endometrial scratching did not result in a higher live birth rates than no intervention in all women undergoing IVF\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. However, these women only received one endometrial scratch at a relatively broad time range of the menstrual cycle that is between day 3 of the preceding cycle to day 3 of the IVF cycle month itself. Further subgroup analysis did not show any benefit in the number of participants who failed implantation at least twice although this was not planned a-priori\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. Twenty-four percent of the participants undergoing a single endometrial scratch comprising 85 and 81 participants had two or three previous unsuccessful embryo transfers respectively. Hence, the small numbers may be underpowered to statistically reflect a fifteen percent difference in live birth rates. In our study, seventeen women (46%) had a clinical pregnancy after performing two sequential endometrial scratches prior to IVF. This is consistent with a recent meta-analysis by Vitagliano et al 2018 which suggested that two luteal phase LEI is the number of interventions that gave a beneficial outcome in women with RIF undergoing IVF\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. A meta-analysis involving 2,537 participants showed an improvement in clinical pregnancy rates for participants with 2 or more failed IVF/ ICSI cycles back in 2019\u003csup\u003e19\u003c/sup\u003e and an extension of it by Hoogenhuijze et al, showed in an individual participant data meta-analysis representing 4,112 participants an improved odds ratio of 1.29 of LBR\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eCurrently, there is no consensus for the definition of RIF due to the clinical heterogenicity of studies performed. Our selection criteria is consistent with a recent systemic review by Polanski et al which defined RIF as the absence of implantation after two consecutive cycles of IVF where the cumulative number of transferred embryos was no less than four for cleavage-stage embryos\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. A uniform and clear definition of RIF is important as it will aid the counselling and expectation of couples undergoing repeated IVF cycles to achieve a successful pregnancy. In addition, it will clearly define the parameters for which LEI improves pregnancy outcomes as what we have observed in this cohort and our previous study\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eThe immune landscape in the endometrium is dynamic with an influx of macrophages, DCs and NK cells during the secretory phase of the menstrual cycle\u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. The low number of CD45\u0026thinsp;+\u0026thinsp;cells in the endometrium in the early follicular to early secretory phase undergoes a five-fold increase during the secretory phase\u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e, peaking at the late secretory phase\u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e. Within the innate immune system, uterine NK (uNK) cells and APCs including macrophages and DCs comprise a large proportion of the decidual leukocyte population and they are known to play important roles in modulating trophoblast invasion, inflammation, angiogenesis and vascular remodelling during implantation\u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e,\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e. Patients with RIF have been shown to have significantly raised NK and B cells\u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e,\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e, suggesting the presence of a potential differential pro-inflammatory bias in RIF patients.\u003c/p\u003e\u003cp\u003eUnravelling the mechanism behind LEI will also be beneficial in explaining the observable benefits of LEI and in this study, we looked into the immune milieu of the endometrial biopsies for answers. LEI induces an inflammatory response that promotes implantation although its exact mechanism is not elucidated\u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e. In our study, we showed that T-cells were the predominant immune cell, followed by NK cells and CD14\u0026thinsp;+\u0026thinsp;macrophages in the endometrium of the cohort of RIF patients. This is congruent with Givan et al where it was established back in 2011 that CD3\u0026thinsp;+\u0026thinsp;T cells constitutes approximately 40% of the CD45\u0026thinsp;+\u0026thinsp;cells in the uterine endometrium in both proliferative and secretory phase and estimated the proportion of NK cells to be around 25% of the CD45\u0026thinsp;+\u0026thinsp;leukocytes\u003csup\u003e44\u003c/sup\u003e. Flynn et al however, found that T cells population vary throughout the menstrual cycle, comprise 40\u0026ndash;60% of the total leukocyte population in the proliferative phase and drops to \u0026lt;\u0026thinsp;10% in the late luteal phase in healthy women\u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. T cells are activated by the innate immune system and are effector cells of the adaptive immune system that affect the activity of other immune cells through secreted cytokines that mount an immune response against pathogens\u003csup\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e. Our study showed instead that T cells were the predominant immune cell population in the luteal phase in our population of women with RIF. Interestingly, within the non-pregnant population, there is a further and significant increase in the T cell population after the LEI, possibly indicating further increased inflammation in the endometrium. On the contrary, that was not observed in the pregnant group. This observation is congruent with Ganeva and colleagues\u0026rsquo; findings where there were significantly lower CD3\u0026thinsp;+\u0026thinsp;T cells in RIF women with a successful implantation than those who were unsuccessful \u003csup\u003e\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e. However, comparison of the T cell population in the 2nd LEI sample between the pregnant and non-pregnant group in our study did not reveal any differences. This could be due to the large inter-patient variability, with a small sample size of 20 in the non-pregnant and 17 in the pregnant group. Nonetheless, our data along with others\u003csup\u003e\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e suggest that the LEI could have set off another mechanism to inhibit the T cell from further increase in women who respond positively.\u003c/p\u003e\u003cp\u003eDecidual NK cells contribute to pregnancy by increasing the blood flow through remodeling of maternal arterioles at the feto-maternal interface and helping the migration of the trophoblast. NK cells is also responsible for the secretion of angiogenic factors such as vascular endothelial growth factor and angiopoietin-2, as well as cytokines and growth factors such as TNF-α, IL-10, GM-CSF, placental growth factor, IL-1β, TGF-β1, CSF-1, LIF, and IFN-γ\u003csup\u003e\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e. Therefore, their distribution and activation at the time of fertilization and implantation plays a critical role in pregnancy outcome\u003csup\u003e\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e. NK cells account for 20% of lymphocytes in the proliferative phase and increases to 40\u0026ndash;50% in the luteal phase and a maximum of 70\u0026ndash;80% during decidualization\u003csup\u003e\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e,\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e. In contrast, NK cells represent approximately 26\u0026ndash;29% of CD45\u0026thinsp;+\u0026thinsp;leukocytes in our RIF population, as opposed to the nearly 70% of endometrial leucocytes in the late luteal phase or early pregnancy\u003csup\u003e\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e,\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u003c/sup\u003e. This corroborated with other studies on women with RIF\u003csup\u003e\u003cspan additionalcitationids=\"CR54\" citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e\u003c/sup\u003e and corresponds well with the postulation that women with subfertility may have lower uNK cells compared to healthy women. From our results, LEI does not alter the proportion of NK cells in women with RIF. NK cell subpopulations are identified by differential expression of a range of NK receptors\u003csup\u003e\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e\u003c/sup\u003e whereby circulating NK cells are primarily CD56\u003csup\u003edim\u003c/sup\u003e CD16\u003csup\u003ebright\u003c/sup\u003e whereas uNK cells are mostly CD56\u003csup\u003ebright\u003c/sup\u003e CD16\u003csup\u003edim \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e\u003c/sup\u003e. However, we did not distinguish this NK subpopulation which is a limitation of our study.\u003c/p\u003e\u003cp\u003eThe next most abundant cell types are B cells, which are known to be found throughout the cycle, at low levels, corroborating with our findings of approximately 2% of the CD45\u0026thinsp;+\u0026thinsp;cell population\u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e,\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e\u003c/sup\u003e. With their low levels, they have long been perceived as insignificant until more recently where they are shown to have distinct characteristics and may be actively involved in shaping the endometrial immune environment\u003csup\u003e\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e\u003c/sup\u003e. The quantity of B cells within the endometrium is altered in certain pathologies in presence of endometrial inflammation\u003csup\u003e\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e\u003c/sup\u003e. There are conflicting findings as to whether RIF patients have increased B-cell numbers\u003csup\u003e\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e\u003c/sup\u003e. In our study, within our definition of RIF patients, we showed an increase of 1.2% (50%) increase in the proportion of B cells within the entire cohort though non statistically significant. We observed a similar increase with B cells in both the non-pregnant and pregnant cohort (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE), suggesting an inflammatory response with the LEI. A wide patient heterogeneity as well as a small sample size could have attributed to the statistical significance and more should be done to look at this.\u003c/p\u003e\u003cp\u003eDendritic cells are APCs that process and present antigenic materials to T lymphocytes\u003csup\u003e\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e,\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e\u003c/sup\u003e and bridge the innate and adaptive immune systems\u003csup\u003e\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e,\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e\u003c/sup\u003e. We noted a trend towards a reduction of both CD141\u0026thinsp;+\u0026thinsp;DCs and CD1c\u0026thinsp;+\u0026thinsp;DCs though non statistically significant. Both CD141\u0026thinsp;+\u0026thinsp;and CD1c\u0026thinsp;+\u0026thinsp;DCs have a common origin of myeloid precursors and produces pro-inflammatory IL-1B, 6, 8 and TNF\u003csup\u003e\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e\u003c/sup\u003e. They are also responsible for taking up, processing and presenting protein antigens to CD4\u0026thinsp;+\u0026thinsp;and CD8\u0026thinsp;+\u0026thinsp;T cells\u003csup\u003e\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e\u003c/sup\u003e. During pregnancy, the maternal-fetal interface undergoes dynamic changes to allow the fetus to grow and develop in the uterus, despite being recognized by the maternal immune cells. Our observation of the reduction in both DCs subtypes following an LEI may not have an impact difference as it is statistically non-significant but if that is due to the power of the study, this reduction could mean aiding in immune tolerance toward the semi-allogeneic fetus, contributing to the beneficial effects of LEI for women with RIF. Analysis of rare cells in the endometrium has its own set of challenges of weighing between the scientific benefits provided by a large sample volume versus the clinical and ethical difficulties of obtaining tissue\u003csup\u003e\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e\u003c/sup\u003e. A larger sample size, or the use of newer high resolution technologies like single cell analyses may be required to illuminate this field.\u003c/p\u003e\u003cp\u003eOverall, we did not find any significant changes of immune cell subtypes before and after LEI in subsequent luteal phase cycles in women with RIF. No significant differences were observed in DCs, macrophages, NK cells and B cells between the first and second Pipelle biopsy which is congruent with Ganeva at el where there were non-significant differences in median of percentages of T cells, NK cells, macrophages, monocytes and B cells\u003csup\u003e\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e. Of particular interest, both their and our study showed a significant change in the T cells, although our participants of interest are different, theirs being women with RIF with and without a successful implantation while ours are women with RIF undergoing LEI without and without getting pregnant\u003csup\u003e\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e. The immune landscape is different in women with RIF as compared to normal women. Endometrial immune dysregulation is also implicated in women with different pathologies including recurrent pregnancy loss, preterm labour and endometriosis \u003csup\u003e\u003cspan additionalcitationids=\"CR67\" citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e\u003c/sup\u003e. In our study, twenty one (57%) and five women (14%) had unexplained fertility and endometriosis respectively. The peripheral and endometrial immune system are altered in women with endometriosis with macrophages, immature DCs and regulatory T cells behaving differently \u003csup\u003e\u003cspan additionalcitationids=\"CR69\" citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e\u003c/sup\u003e and that can confound our results, exacerbated by the small samples size.\u003c/p\u003e\u003cp\u003eImmune activation or imbalance between immune effectors and regulators has been reported in the patients with reproductive failure hence the importance of this study\u003csup\u003e\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u003c/sup\u003e. That said, the etiology of RIF is likely multifactorial and thus, the mechanism(s) for an improvement in clinical pregnancy rates brought about by LEI may not only be immunologically based or even if it is, may not be directly measured through the differential proportion of the cells types we looked at. The mechanical abrasion followed by wound healing may positively influence implantation rates by altering the release of cytokines, chemokines, IL-15, adhesion molecules, growth factors and hormones\u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e or switching on and/or activating dormant genes\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e,\u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e\u003c/sup\u003e. As the human endometrium undergoes more than 400 cycles of regeneration, differentiation and shedding during a women's reproductive life span\u003csup\u003e\u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e\u003c/sup\u003e, it is also feasible that menstruation may bestow plasticity on the uterus through activation of endometrial stem cells. This may be timely for tissue remodelling associated with deep placentation and enhancing embryonic implantation for pregnancy\u003csup\u003e\u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e73\u003c/span\u003e\u003c/sup\u003e. We have previously shown that the decidual response is seen at the embryo-maternal interface with differentiating perivascular stem cells establishing distinct cytokine and chemokine profiles that affect local immune responses in pregnancy\u003csup\u003e\u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e74\u003c/span\u003e\u003c/sup\u003e. We have also shown that endometrial mesenchymal stem/ progenitor cells derived from the second LEI tend to be more proliferative with a shorter doubling time, with a reduced decidualization propensity\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eThe novelty of our study includes the approach to LEI involving the same patient over consecutive menstrual cycles where the use of uterine catheter acts as intervention while concurrently allows tissue collection. Patients also act as their own control which can reduce inter-patient variability that can confound results. In addition, we employed a validated assay (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) that can interrogate the composition and content of the various immune cell subtypes with high specificity and sensitivity, circumventing the limitations of traditional methods analysis\u003csup\u003e\u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e75\u003c/span\u003e,\u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e76\u003c/span\u003e\u003c/sup\u003e. The approach of treating endometrial specimens as a cell suspension is a powerful approach as this technique has significant advantages over the traditional immuno-histochemical staining methods, including the ability to precisely interrogate multiple markers simultaneously from a single specimen and exclude false positive or negative signals from non-viable cells. That said, limitations of our study include a small sample size of fifty-three women with a high drop-out rate of sixteen patients (30%) and that our results are dependent on the Pipelle biopsies obtained which are beset by different sampling regions between patients as well as between the first and second LEI. In addition, we did not perform subset analysis of the NK (uterine and non -uterine) and T cells. T cell subsets such as CD4, CD8 and T regulatory cells and their activation status which are implicated in patients with subfertility can further ascertain better understanding on the complex interactions between the innate and adaptive immune system during implantation and pregnancy.\u003c/p\u003e\u003cp\u003eIn this small but well-defined RIF population where sequential paired samples were analysed by high resolution flow cytometry, the immune landscape had remained largely unchanged post LEI in women with RIF. Further research in this area, including uncovering the immune activation statuses of NK and T-cells is needed. These can be done by transcriptomics, applying RNA sequencing for gene expression analysis in a single cell manner and/or high-resolution cytometry by time-of-flight (CYTOF). Elucidating the critical pathways surrounding the beneficial effects of LEI in RIF patients will not only offer a deeper understanding of the critical events surrounding human implantation but opens the possibility of devising approaches to enhance fertility outcomes.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003eEthics Approval\u003c/h2\u003e\u003cp\u003e The current study was reviewed and approved by the SingHealth Centralized Institutional Review Board (CIRB 2013/215/D). Informed written consent was obtained for all participants before their participation in the study at KKIVF Centre, KK Women\u0026rsquo;s and Children\u0026rsquo;s Hospital. All experimental procedures were performed in accordance with the relevant guidelines and regulations.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eInclusion and Exclusion Criteria\u003c/h3\u003e\n\u003cp\u003eOur inclusion criteria are women with RIF undergoing IVF with RIF defined as failure to achieve a clinical pregnancy after a transfer of minimum four good-quality embryos in a minimum of two or more previous failed embryo transfers. Other inclusion factors include women\u0026thinsp;\u0026lt;\u0026thinsp;40 years of age with primary subfertility with normal ovarian reserves, good response to ovarian stimulation and optimal luteal endometrial thickness\u0026thinsp;\u0026gt;\u0026thinsp;7 mm at embryo transfer and a normal hormonal profile. Our exclusion criteria included women\u0026thinsp;\u0026gt;\u0026thinsp;40 years old, BMI\u0026thinsp;\u0026gt;\u0026thinsp;35 and women who do not meet our criteria for RIF or women who declined a repeat endometrial scratch in the following menstrual cycle. Serum progesterone was measured to check for ovulatory status. Serum beta human chorionic gonadotrophin (βhCG) was measured 17 days after IVF embryo transfer to determine if women became pregnant with a positive βhCG result\u0026thinsp;\u0026gt;\u0026thinsp;25 mIU per milliliter. Clinical pregnancy rate was defined as the presence of intrauterine gestation sac on ultrasound scan 2 weeks after a positive βhCG result.\u003c/p\u003e\n\u003ch3\u003eSample Collection and Processing\u003c/h3\u003e\n\u003cp\u003eTo characterize the immunological landscape alterations in LEI, we performed sequential implantation-phase (mid-luteal) endometrial biopsies (first and second biopsies) in RIF patients prior to IVF. These women underwent two consecutive LEI with a Pipelle endometrial catheter to obtain endometrial tissue during the first and succeeding mid-luteal phase (Day 21\u0026ndash;25) of their menstrual cycles (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The endometrial scratch procedure was performed by clinic doctors using a Pipelle, a plastic biopsy catheter approximately 3 mm in diameter (Pipelle de cornier, Laboratoire CCD, France).\u003c/p\u003e\u003cp\u003eEndometrial tissues obtained were minced into fragments and digested for 30 min at 37\u0026deg;C with 1 mg/mL of collagenase type IV (Sigma-Aldrich, St Louis, MO), 0.2 mg/mL of DNase (Roche Applied Science, Germany) and 1 mg/mL of bovine serum albumin in PBS. The single cell suspension was passed through a mesh, washed in PBS, treated with Ammonium-Chloride-Potassium lysing buffer (0.01 M KHCO3 buffer, 0.16M NH4Cl and 0.1 mM ethylenediaminetetraacetic acid (EDTA) to remove red blood cells, washed and resuspended in PBS as previously described\u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eFlow Cytometry\u003c/h2\u003e\u003cp\u003eCells were blocked in human blocking buffer (5% human serum (Sigma-Aldrich), 1% rat serum (Sigma-Aldrich), 1% mouse serum (Sigma-Aldrich), 5% FBS, and 2 mM EDTA) for 15 min at 4\u0026deg;C and incubated with antibody cocktails (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) for 30 min at 4\u0026deg;C. Cell suspensions were stained for viability with 1:3,000 DAPI (Sigma-Aldrich). Flow cytometric analyses were performed on an LSR II (Becton Dickinson) and data analysed with FlowJo software (TreeStar). Two antibody cocktails were used. Live cells (DAPI\u003csup\u003e\u0026minus;\u003c/sup\u003e) were first selected. Doublets were then removed and leukocytes (CD45\u003csup\u003e+\u003c/sup\u003e) identified. With the first antibody cocktail, tissue CD14\u003csup\u003e+\u003c/sup\u003e macrophages, pDCs, CD141\u003csup\u003e+\u003c/sup\u003e DCs, CD1c\u003csup\u003e+\u003c/sup\u003e DCs were identified (Fig.\u0026nbsp;2Ai). Antigen presenting cells were identified as HLA-DR\u003csup\u003e+\u003c/sup\u003e and lineage (CD3, CD19, CD20)\u003csup\u003e\u0026minus;\u003c/sup\u003e. CD14\u003csup\u003e+\u003c/sup\u003e macrophages which also express the macrophage markers CD163 and CD64 were enumerated. From the CD14-CD16- fraction, pDCs were identified by CD123\u003csup\u003e+\u003c/sup\u003e and CD11c\u003csup\u003e\u0026minus;\u003c/sup\u003e. From the CD123\u003csup\u003e\u0026minus;\u003c/sup\u003eCD11c\u003csup\u003e\u0026minus;\u003c/sup\u003e population, while CD141\u003csup\u003e+\u003c/sup\u003eCD1c\u003csup\u003e\u0026minus;\u003c/sup\u003e and CD1c\u003csup\u003e+\u003c/sup\u003eCD141\u003csup\u003e\u0026minus;\u003c/sup\u003e were enumerated. With the second antibody cocktail, CD3\u003csup\u003e+\u003c/sup\u003e T cells, CD19\u003csup\u003e+\u003c/sup\u003eCD20\u003csup\u003e+\u003c/sup\u003e B cells and CD56\u003csup\u003e+\u003c/sup\u003e NK cells were identified and enumerated (Fig.\u0026nbsp;2Aii).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\u003ch2\u003eStatistical Analysis\u003c/h2\u003e\u003cp\u003eThe data is presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation. Statistical analysis comparing first and second LEI was performed using non-parametric (Mann-Whitney for unpaired or Wilcoxon signed-ranked test for paired data) or Student\u0026rsquo;s t-test where appropriate with Graphpad Prism 10. A two-sided p-value at the 5% level was considered to indicate statistical significance.\u003c/p\u003e\u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cb\u003eFunding Declarations\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThis study was supported by the KK Women\u0026rsquo;s and Children\u0026rsquo;s Hospital Health Endowment Fund (KKHHEF/2013/07) and National Medical Research council (NMRC) seed fund (0004/2017). JKYC is supported by National Medical Research Council (CIRG/1484/2018, NMRC CSA (SI)/008/2016, CIRG21jun-0045 and STaR22jul-0004).\u003c/p\u003e\u003cp\u003e\u003ch2\u003eAdditional Information\u003c/h2\u003e\u003cp\u003eThe authors declare no conflicts of interest.\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eRWKL, NM, FG, YHL and JKYC study conceptualization. AM and NM designed and performed the flow experiments. RWKL, YF, YHL analysed the data and drafted the paper. RWKL, TYT, JKYC obtained funding, consented patients and collected data. All authors read, reviewed and approved the final version of the article.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eThe authors wish to thank Dr Sadhana Nadarajah, Dr Tan Heng Hao, Miss Ng Xiang Wen from KK Women\u0026rsquo;s and Children\u0026rsquo;s Hospital for clinical administrative support, Mr Gurmit Singh from Singapore Immunology Network (SIgN) for tissue sample processing and flow cytometry analysis.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe authors confirm that the data supporting the findings are available within the article. Please contact the corresponding author for more information.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eLuke, B. et al. Cumulative birth rates with linked assisted reproductive technology cycles. \u003cem\u003eN Engl. J. 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During the implantation period, modulation in immune milieu occur in tandem with profound morphologic and functional changes in the endometrium. The landscape of immune cells in the endometrium in pre- and post-LEI in RIF is currently unknown. Thirty-seven women with RIF (age 34.6 ± 3.3 years old) underwent LEI by two sequential mid-luteal phase endometrial biopsies prior to embryo transfer. To characterize the immunological landscape alterations in LEI, we performed immunophenotypic assessment with flow cytometry to provide insights into the basal (first biopsy) and altered (second biopsy) biology of dendritic cells (DC), macrophages, natural killer (NK), T and B cells in the RIF population before and after LEI. Clinical pregnancies occurred in seventeen women (46%). Among analysed immune cells, T (34.6%) and NK cells (26.2%) predominate in the mid-luteal endometrium. A consistent increase in lymphocytes and decrease in antigen presenting cells (APCs) were observed between the two biopsies although not statistically significant. Segregating by pregnancy outcomes demonstrated a significant increase in the T cells in the women who did not get pregnant post the local endometrial injury which was not observed in the group of women with RIF who fell pregnant (p = 0.03). There were no further difference in any of the other measured immune cell subsets between the first and second endometrial biopsy. We found limited changes in the immune cell compartments after LEI. Further research with higher resolution methods may provide more information on the effects of LEI.\u003c/p\u003e","manuscriptTitle":"The Immune Milieu After Local Endometrial Injury in Women with Recurrent Implantation Failure","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-16 19:20:53","doi":"10.21203/rs.3.rs-7494176/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-11-10T06:52:47+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-11-09T09:40:05+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"259792148429534818827378752596045770623","date":"2025-10-23T07:25:37+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"290812013872811398632811092058393570953","date":"2025-10-23T05:58:13+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-13T11:02:00+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"55845432523922424160658178729963001667","date":"2025-10-03T10:29:58+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-10-03T02:12:10+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-10-03T01:55:45+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-09-23T10:55:30+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-09-03T00:29:18+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2025-09-03T00:25:24+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"57ea1a19-3c7a-4a26-80d2-7593ee27d8e6","owner":[],"postedDate":"October 16th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":56214123,"name":"Health sciences/Diseases"},{"id":56214124,"name":"Biological sciences/Immunology"},{"id":56214125,"name":"Health sciences/Medical research"}],"tags":[],"updatedAt":"2026-01-05T16:01:36+00:00","versionOfRecord":{"articleIdentity":"rs-7494176","link":"https://doi.org/10.1038/s41598-025-34198-7","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2025-12-30 15:58:13","publishedOnDateReadable":"December 30th, 2025"},"versionCreatedAt":"2025-10-16 19:20:53","video":"","vorDoi":"10.1038/s41598-025-34198-7","vorDoiUrl":"https://doi.org/10.1038/s41598-025-34198-7","workflowStages":[]},"version":"v1","identity":"rs-7494176","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7494176","identity":"rs-7494176","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}