Effects of Nonablative Er‐YAG Laser on Human Endometrial Stromal Cells (hESCs): A Pilot Study

The study was approved by the institutional review board and was performed in accordance with the ethical standards as presented in the 1964 Declaration of Helsinki.

There was statistically significant difference in median MMP‐2 levels at 12 h in hESC cultures ( p  = 0.012) (Table  1 and Figure  1 ). The application of Er‐YAG laser increased median MMP‐2 levels from a basal level of 12.17 (0–16.10) pg/ml/µg protein to 60.22 (50.61–76.77) pg/ml/µg protein, indicating a fivefold increase (Table  1 ). In the E 2 ‐only group, there was nearly a 17‐fold increase, while the addition of P4 produced an additive response (Figure  1 ). Er‐YAG laser application affected the median levels of MMP‐2 in the conditioned medium of hESCs at 24 h ( p  = 0.045). At 24 h, median MMP‐2 levels rose by 10‐fold in the E 2 ‐only group and fivefold in the E 2  + P 4 group after Er‐YAG laser application ( p  = 0.045) (Figure  1 ). The Er‐YAG laser application significantly upregulated median MMP‐2 levels in both the E 2 ‐only and E 2  + P 4 groups at 48 h ( p  = 0.021) (Figure  1 ), with significant differences observed until the 72nd hour ( p  = 0.009). Compared with the basal MMP‐2 output in cultures incubated with E 2 alone (15.65 [2.10–18.66 pg/mL/µg protein]), median MMP‐2 output increased to 44.13 (29.80–61.56) pg/mL/µg protein with Er‐YAG laser application in 72 h, although this was statistically nonsignificant. In the E 2  + P 4 treated cells, Er‐YAG laser application increased median MMP‐2 output by ninefold from 4.64 (0–9.03) pg/ml/µg protein to 41.06.(30.31–69.92) pg/mL/µg protein (Figure  1 ). After 72 h of Er‐YAG laser application, median MMP‐2 levels in hESCs alone were markedly upregulated, from 20.73 (13.08–20.82) pg/ml/µg protein to 62.28 (62.08–71.02) pg/mL/µg protein. The ELISA results of MMP2, TNF‐ɑ, IL‐6, VEGF‐A, and IGFBP1 for all experimental groups. Note: Kruskal–Wallis test ( † p value calculated to compare the measurements of the first five experimental groups; ‡ p value calculated to compare the measurements of seven groups; c p calculated for measurements over time.). Abbreviations: E 2 , estradiol; ELISA, enzyme‐linked immunosorbent assay; hESC, human endometrial stromal cell; IGFBP‐1, insulin‐like growth factor‐binding protein‐1; IL‐6, interleukin‐6; L, laser; MMP2, matrix metalloproteinase‐2; P 4 , progesterone; TNF‐α, tumor necrosis factor‐alpha; VEGF‐A, vascular endothelial growth factor‐A. The boxplots of the MMP‐2 levels in the cultured media of human endometrial stromal cells (hESCs). Confluent hESC cultures were treated with E 2 (10 −8  M) (group E 2 ), E 2 (10 −8  M)+ ethanol (0.1%) (group E 2  + S), E 2 (10 −8  M) + P 4 (10 −7  M) (group E 2  + P 4 ), Er‐YAG laser + E 2 (10 −8  M) (group E 2  + L), Er‐YAG laser+ E 2 (10 −8  M) + P 4 (10 −7  M) (group E 2  + P 4  + L) for 12, 24, 48, and 72 h. Only hESCs (incubated in serum‐free DMEM/F12) and hESCs + Er‐YAG laser (hESCs + L) groups were incubated for 12 and 72 h. MMP‐2 levels were quantified by ELISA in culture media and normalized to total cell protein ( n  = 3, median (Q1–Q3). DMEM, Dulbecco modified Eagle medium; E 2 , estradiol; ELISA, enzyme‐linked immunosorbent assay; Er‐YAG, nonablative Erbium YAG; ESC, endometrial stromal cell; MMP‐2, matrix metalloproteinase‐2; P 4 , progesterone. Overall, the remodeling effect of the Er‐YAG laser on MMP‐2 persisted for 72 h, but no time‐dependent differences were observed in the conditioned media across treatments. The immunomodulatory effect of the Er‐YAG laser based on Th1 cell activity was assessed through TNF‐α levels. No TNF‐α activity was detected in ELISA tests after 12 h, except in the E 2  + S and E 2  + L groups (Figure  2 ). Median TNF‐α levels began to change at 24 h, with significant differences observed at 48 h ( p  = 0.029), where levels were lower in the Er‐YAG laser groups ( p  = 0.029) (Table  1 ). This downregulation persisted for up to 72 h ( p  = 0.019) (Table  1 ). The Er‐YAG laser significantly downregulated median TNF‐α levels from 774.74 (677.93–867.64) pg/mL/µg protein to 0 (0–390.05) pg/ml/µg protein when applied to E 2  + P 4 group, from 632.02 (503.04–1207.16) pg/mL/µg protein to 204.14 (105.38–353.72) pg/mL/µg protein when applied to E 2 ‐only group, and from 794.17 (683.01–857.54) pg/mL/µg protein to 123.02 (97.61–277.57) pg/mL/µg protein when applied directly on hESC (Table  1 ). The boxplots of the TNF‐α and IL‐6 levels in the cultured media of human endometrial stromal cells (hESCs). Confluent hESC cultures were treated with E 2 (10 −8  M) (group E 2 ), E 2 (10 −8  M) + ethanol (0.1%) (group E 2  + S), E 2 (10 −8  M) + P 4 (10 −7  M) (group E 2  + P 4 ), Er‐YAG laser + E 2 (10 −8  M) (group E 2  + L), Er‐YAG laser+ E 2 (10 −8  M) + P 4 (10 −7  M) (group E 2  + P 4  + L) for 12, 24, 48, and 72 h. Only hESCs (incubated in serum‐free DMEM/F12) and hESCs + Er‐YAG laser (hESCs + L) groups were incubated for 12 and 72 h. (A) represents subsequent time points for TNF‐α analysis, and (B) represents subsequent time points for IL‐6 analysis. TNF‐α and IL‐6 levels were quantified by ELISA in culture media and normalized to total cell protein ( n  = 3, median (Q1–Q3). DMEM, Dulbecco modified Eagle medium; E 2 , estradiol; ELISA, enzyme‐linked immunosorbent assay; Er‐YAG, nonablative Erbium YAG; ESC, endometrial stromal cell; IL‐6: interleukin‐6; MMP‐2, matrix metalloproteinase‐2; P 4 , progesterone; TNF‐α, tumor necrosis factor‐alpha. Median TNF‐α levels significantly increased over time in the E 2 , E 2  + S or E 2  + P 4 groups, while remaining stable in the Er‐YAG laser‐treated groups(E 2  + L and E 2  + P 4  + L) (Table  1 ). The immunomodulatory effect of the Er‐YAG laser on Th2 cell activity was measured by IL‐6 levels. At 12 h, median IL‐6 levels varied significantly among groups ( p  = 0.011) (Table  1 ), with the Er‐YAG laser increasing IL‐6 by about 20‐fold in E 2 ‐alone, 2.4‐fold in the E 2  + P 4 groups and 3.5‐fold when applied directly to hESCs (Figure  2 and Table  1 ). By 24 h, IL‐6 levels in laser‐treated media remained elevated but without statistical significance ( p  = 0.134) (Table  1 ). At 48 and 72 h, IL‐6 levels were similar between control and laser‐treated groups ( p  = 0.717, p  = 0.609) (Table  1 ). The impact of the Er‐YAG laser on neoangiogenesis was assessed by measuring VEGF‐A levels. At 12 h, there was no statistically significant difference in median VEGF‐A levels between the groups with or without laser treatment ( p  = 0.201) (Table  1 ). Median VEGF‐A levels increased more than ninefold 12 h after Er‐YAG laser application in the media of hESCs incubated with E 2 alone, although statistically nonsignificant (Figure  3 ). After 24 h, the media of hESCs treated with Er‐YAG laser showed lower median values of VEGF‐A compared to their controls, however statistically nonsignificant ( p  = 0.076) (Table  1 ). After 48 h of Er‐YAG laser application, median VEGF‐A levels were downregulated, although statistically nonsignificant (Table  1 ). At the 72 h, the lower median VEGF‐A values persisted in the media of hESCs incubated with E 2 , E 2  + S, E 2  + P 4 or hESCs alone treated with the Er‐YAG laser ( p  = 0.021) (Table  1 ). The boxplots of the VEGF‐A levels in the cultured media of human endometrial stromal cells (hESCs). Confluent hESC cultures were treated with E 2 (10 −8  M) (group E 2 ), E 2 (10 −8  M)+ ethanol (0.1%) (group E 2  + S), E 2 (10 −8  M) + P 4 (10 −7  M) (group E 2  + P 4 ), Er‐YAG laser + E 2 (10 −8  M) (group E 2  + L), Er‐YAG laser+ E 2 (10 −8  M) + P 4 (10 −7  M) (group E 2  + P 4  + L) for 12, 24, 48, and 72 h. Only hESCs (incubated in serum‐free DMEM/F12) and hESCs + Er‐YAG laser (hESCs + L) groups were incubated for 12 and 72 h. VEGF‐A levels were quantified by ELISA in culture media and normalized to total cell protein ( n  = 3, median (Q1–Q3). DMEM, Dulbecco modified Eagle medium; E 2 , estradiol; ELISA, enzyme‐linked immunosorbent assay; Er‐YAG, nonablative Erbium YAG; ESC, endometrial stromal cell; P 4 , progesterone; VEGF‐A, vascular endothelial growth factor‐A. A significant time‐dependent increase in VEGF‐A levels was observed in the media of hESCs incubated with E 2 ( p  = 0.033) and E 2  + P 4 ( p  = 0.032). The impact of the Er‐YAG laser on decidualization of the endometrium was assessed by measuring the level of IGFBP‐1 secreted by the cultured hESCs. IGFBP‐1 levels were comparable between conditioned media of hESCs regardless of Er‐YAG laser application at the 12th hour mark ( p  = 0.165) (Table  1 ). A statistically significant difference was observed in median IGFBP‐1 levels between the media at 24 h, with higher levels of IGFBP‐1 in the groups that received Er‐YAG laser ( p  = 0.041). The median IGFBP‐1 increased approximately twofold after 24 h by Er‐YAG laser application in media incubated with E 2 (Figure  4 ). The boxplots of the IGFBP‐1 secretions in the cultured media of human endometrial stromal cells (hESCs). Confluent hESC cultures were treated with E 2 (10 −8  M) (group E 2 ), E 2 (10 −8  M) + ethanol (0.1%) (group E 2  + S), E 2 (10 −8  M) + P 4 (10 −7  M) (group E 2  + P 4 ), Er‐YAG laser+ E 2 (10 −8  M) (group E 2  + L), Er‐YAG laser + E 2 (10 −8  M) + P 4 (10 −7  M) (group E 2  + P 4  + L) for 12, 24, 48, and 72 h. Only hESCs (incubated in serum‐free DMEM/F12) and hESCs + Er‐YAG laser (hESCs + L) groups were incubated for 12 and 72 h. IGFBP‐1 levels were quantified by ELISA in culture media and normalized to total cell protein ( n  = 3, median (Q1–Q3). DMEM, Dulbecco modified Eagle medium; E 2 , estradiol; ELISA, enzyme‐linked immunosorbent assay; Er‐YAG, nonablative Erbium YAG; ESC, endometrial stromal cell; IGFBP‐1, insulin‐like growth factor‐binding protein‐1; P 4 , progesterone. The changes in IGFBP‐1 levels following Er‐YAG laser application did not persist beyond 24 h, with all groups showing similar IGFBP‐1 levels at both the 48th hour and 72nd hour upon measurements ( p  = 0.402 and p  = 376, respectively) (Table  1 ). No time‐dependent variation was observed in median IGFBP‐1 levels in the conditioned media of cultured hESCs, regardless of whether they were incubated with E 2 , or E 2  + P 4 , and irrespective of whether they received Er‐YAG laser (Table  1 ).

This pilot study is the first to investigate the effects of nonablative Er‐YAG laser on hESCs. Our findings show that the laser induces tissue remodeling via MMP‐2, enhances Th2 activity through IL‐6 while suppressing Th1 activity by decreasing TNF‐α, and positively affects decidualization. However, its impact on neoangiogenesis in the endometrium within 72 h is less pronounced. These findings may help elucidate the pathophysiology of recurrent implantation failure, as well as other human implantation disorders such as recurrent pregnancy loss, pre‐eclampsia, and intrauterine growth restriction, offering a potential novel treatment approach. In this study, we found that non‐ablative Er‐YAG laser application increased MMP‐2 levels in hESCs. Proteinases, including MMPs, facilitate extracellular matrix degradation necessary for placental villi formation during the invasion stage [ 23 ]. Nonablative Er‐YAG laser stimulates the production and secretion of HSP in the target tissue, with peak activity in the endometrium during the window of implantation (WOI), when receptivity is highest [ 14 , 24 ]. Estrogen and progesterone regulate HSP synthesis, enhancing endometrial receptivity [ 25 ]. The involvement of HSPs in MMP‐2 activation highlights their role in decidualization, implantation, and placentation [ 25 , 26 , 27 ]. Thus, non‐ablative Er‐YAG laser may benefit the endometrial receptivity during the WOI by increasing MMP‐2 levels, similar to the effects of estrogen and progesterone. Our pilot study revealed that non‐ablative Er‐YAG laser has opposing effects on TNF‐α and IL‐6 production in human ESCs, suggesting its potential for endometrial immune modulation. We demonstrated that the non‐ablative Er‐YAG laser decreased TNF‐α levels, especially at 24 h, with this effect persisting up to 72 h. In contrast, IL‐6 production increased at 12 and 24 h but declined after 48 h. This pattern aligns with the known effects of progesterone, which reduces Th1 cytokines like TNF‐α while promoting Th2 cytokines such as IL‐6 within the endometrium [ 28 , 29 ]. Another important impact of progesterone is the increase in body temperature after ovulation [ 30 ]. Additionally, progesterone's thermogenic effect after ovulation may modulate inflammatory markers in the endometrium. In this regard, the non‐ablative Er‐YAG laser may induce heating within the endometrium, thereby modulating the release of inflammatory markers by immune cells within the endometrial stroma [ 11 , 14 ]. By mimicking the effects of progesterone, the Er‐YAG laser may offer hope for patients with luteal phase deficiency and enhance their implantation potential [ 31 , 32 ]. However, further studies are necessary. Additionally, the non‐ablative nature of the Er‐YAG laser contrasts with endometrial scratching, which induces local damage and promotes decidualization through tissue proliferation and immune cell accumulation during healing [ 33 , 34 , 35 , 36 ]. Nonablative Er‐YAG laser provides controlled heating to vaginal mucosal tissue, typically within 60°C–70°C, inducing collagen fibril contraction in deeper layers without causing collagen denaturation or overheating the surface [ 10 , 11 ]. After tissue temperature decreases, it stimulates fibroblast generation from stem cells (fibrocytes), leading to collagen remodeling and neocollagenesis [ 11 ]. Laser therapies have been shown to reduce inflammation and pain while accelerating tissue healing, evidenced by increased epithelial thickness and capillary volume density in treated women. [ 12 , 13 ]. This treatment also regulates cellular proliferation, migration, and growth factor synthesis, while enhancing HSP production during wound healing. Additionally, HSP70 coordinates TGF‐β expression, which is crucial for collagen generation [ 14 ]. These thermochemical effects of Er‐YAG laser may enhance implantation by mimicking progesterone's thermogenic effects and modulating the endometrial immune response, as confirmed by our findings. Several studies suggest that non‐ablative Er‐YAG laser treatment increases VEGF‐A expression, promoting neo‐angiogenesis in various tissues [ 13 , 15 , 37 , 38 ]. However, our study found a decrease in VEGF‐A levels at 24‐, 48‐, and 72‐h posttreatment. This indicates that both immediate and long‐term responses to non‐ablative Er‐YAG laser may differ in terms of VEGF‐A expression [ 39 ]. Additionally, changes in IGFBP‐1 expression may take time to manifest after Er‐YAG laser irradiation, similar to how progesterone stimulates differentiation and decidual marker expression, including IGFBP‐1, after 12 days of incubation [ 40 ]. Further in vivo studies are necessary to elaborate the long‐term effect of Er‐YAG laser application on endometrial tissue. However, it is preliminarily hypothesized that both progesterone and Er‐YAG laser share a common mechanism of action by increasing temperature. The primary strength of this study is its pioneering nature, as the first investigation on this topic. However, it has several weaknesses, including a small sample size and reliance on nonparametric statistical tests, as it is a pilot study. Despite these limitations, the results are promising, highlighting the need for larger studies with a higher number of cultured hESCs to obtain more robust findings. Further research, particularly in vivo studies using animal models followed by human subjects, is necessary to observe the effects of Er‐YAG laser on the endometrium and compare them with in vitro results. The present study may offer a potential treatment for certain clinical problems associated with repeated IVF failure due to the endometrial factors, repeated implantation failure, recurrent early pregnancy loss, pre‐eclampsia [ 41 , 42 , 43 ]. The challenges of thin endometrium and intrauterine adhesions remain significant issues in reproductive medicine [ 44 ]. Application of Er‐YAG laser to endometrial cells/endometrium, as described in this study, may illuminate new treatment options for women experiencing infertility or amenorrhea.

We can conclude that this pilot study would open a new horizon for further laboratory and clinical research on the impact of non‐ablative Er‐YAG laser on human endometrial tissue during the window of implantation. Moreover, the findings of this study may shed light on the pathophysiology of recurrent implantation failure, early pregnancy losses and even endometriosis, suggesting that non‐ablative Er‐YAG laser could be a potential treatment option for these disorders.

In human reproduction, the successful implantation of an embryo into the endometrium depends on several crucial factors. First, the blastocyst must exhibit a high grade of development. Second, the endometrium must be in a receptive state. Third, a complex crosstalk between the blastocyst and the endometrium regulated by various factors is essential for implantation. Under the influence of progesterone, the endometrium undergoes crucial changes that enable implantation. The endometrium is receptive to the blastocyst for a limited period known as the window of implantation (WOI). Noyes was a pioneer in analyzing and establishing criteria for morphological changes in the endometrium through histological dating [ 1 ]. Following these studies, which identified molecular changes in the receptive endometrium during the WOI, recent research has focused on gene profiling. This includes identifying genes that regulate processes such as remodeling, decidualization, and immune modulation during this period, utilizing transcriptomic panels based on next‐generation sequencing [ 2 ]. Various molecules play key roles in the phases of embryo implantation and have been linked to recurrent implantation failure (RIF) [ 2 , 3 , 4 , 5 , 6 , 7 ]. To address RIF, several therapies—including high‐dose progesterone, glucocorticoids, intravenous immunoglobulin, endometrial scratching, and intrauterine infusions of various factors—have been explored. However, due to a lack of evidence‐based support, these treatments are not recommended by several reproductive societies [ 8 , 9 ]. The effects of non‐ablative Erbium YAG (Er‐YAG) laser on the endometrium remain unstudied. The Er‐YAG laser, with a wavelength of 2940 nm, can be set to non‐ablative mode by adjusting the pulse duration. Studies on vaginal mucosa suggest that the controlled thermo‐mechanical and thermo‐chemical effects of the non‐ablative Er‐YAG laser (within an optimal temperature range of 60°C–70°C), induce collagen fibril shrinkage in the deep mucosal layers without causing denaturation or overheating [ 10 , 11 ]. This controlled heat promotes collagen remodeling, neocollagenesis, reduced inflammation, pain relief, and faster healing [ 12 ]. Histologic studies further indicate that non‐ablative Er‐YAG laser may enhance capillary density, stimulate epithelial growth, and increase cellular and protein synthesis, while activating heat shock proteins, particularly HSP70, to support collagen formation [ 13 ]. During the inflammatory response, HSP70 regulates the expression of transforming growth factor‐beta (TGF‐β), a key element in the generation of new collagen and extracellular matrix in the fibrogenic process [ 14 ]. Recent findings suggest that intrauterine exposure to nonablative Er‐YAG laser may stimulate endometrial proliferation while reducing sclerosis of the spiral arterioles in the stroma [ 15 ]. In the same study, intrauterine application of the nonablative Er‐YAG laser significantly enhanced the expression of vascular endothelial growth factor‐A (VEGF‐A), progesterone receptors (PR) and estrogen receptors (ER). In the present study, we aim to investigate the thermo‐chemical effects of nonablative Er‐YAG laser on human endometrial stromal cells (hESCs). These effects may involve mechanisms such as tissue remodeling, immunomodulation, neoangionesis and decidualization. We hypothesize that nonablative Er‐YAG laser may either promote or suppress the secretion of key molecules involved in these processes, thereby influencing implantation.

The authors declare no conflicts of interest.

This study was conducted at Istanbul University‐Cerrahpasa, Cerrahpasa Faculty of Medicine, in the Obstetrics and Gynecology and Histology and Embryology departments. Endometrial biopsy samples were obtained from three fertile women (aged 39–40) in the late proliferative phase, all of whom had regular cycles, had not received hormonal treatment in the past 3 months and were scheduled to undergo hysterectomy or laparoscopy for benign conditions. Samples were placed in Leibovitz‐15 medium (Pan‐Biotech: P04‐27500, Germany) and transported for hESC isolation and culture, before being stored in liquid nitrogen. Written informed consent was obtained from each patient before surgery, in accordance with the 1964 Helsinki Declaration and its later amendments or comparable ethical standards. Ethics committee approval was obtained from the Institutional Clinical Research Ethics Committee (Approval date: 12.07.2023 and Approval number: 732218). No animals or humans were involved in the study. The hESCs were isolated and cultured as a monolayer using the following method: Endometrial tissues were minced with a sterile surgical blade and digested in Hank's‐balanced salt solution containing collagenase B (1 mg/mL, 15 U/mg; Gibco™, 17018029), deoxyribonuclease I (0.1 mg/mL, 1500 U/mg; Sigma‐Aldrich: DN25), penicillin (200 U/mL), and streptomycin (200 mg/mL) for 60 min at 37°C with agitation. The dispersed cells were then filtered through a wire sieve (73 µm‐diameter pore; Sigma‐Aldrich) and cultured in phenol red‐free Dulbecco modified Eagle medium (DMEM)/Ham F‐12 (1:1 vol/vol; Gibco™, 1103902, Thermo Fisher Scientific, UK) supplemented with 10% stripped fetal bovine serum (FBS; Gibco™; 10500064, Brazil) and 1% antibiotics‐antimycotic solution (1:1 vol/vol Gibco™; 15240062). The hESCs were maintained in a standard incubator at 37°C with 95% air/5% CO 2 until they reached 80%–90% confluence. After the second passage, hESCs were plated in matrigel‐coated 35‐mm culture dishes and grown to confluence. They were preincubated with serum‐free DMEM/F12 for 24 h before the experimental treatments. Subsequently, the media from all experimental groups were removed, and cells were washed twice with PBS. The experiment was repeated three times using hESCs from different patients establishing five experimental groups ( n  = 3 each): Estradiol(E 2 )(control), E 2  + Sham (S)(control of E 2  + Progesterone (P 4 )), E 2  + P 4 , E 2  + Laser(L) and E 2  + P 4  + L. The E 2 group served as the control to reflect the physiological environment in women and assess progesterone activity [ 16 ]. Confluent cell cultures adhering to the matrigel‐coated petri dish were incubated in serum‐free DMEM/F12 supplemented with 10 −8  M E 2 (Sigma‐Aldrich; E8875), 10 −8  M E 2  + 0.1% ethanol (as the solvent of P 4 ) and 10 −8  M E 2  + 10 −7  M P 4 (Sigma‐Aldrich: 46412), for the first three groups. In the laser‐treated groups, cells were incubated in serum‐free DMEM/F12 supplemented with either 10 −8  M E 2 or 10 −8 M E2 + 10 −7  M P 4 following the application of non‐ablative Er‐YAG laser. All experimental groups were incubated for 12, 24, 48, and 72 h to evaluate the treatment effects over time. Additionally, two more experimental groups were included at the 12th and 72th hours: one with hESCs incubated in serum‐free DMEM/F12 alone and another (hESCs + L) group incubated in serum‐free DMEM/F12 with non‐ablative Er‐YAG laser application. For both of these additional groups, the incubation period was limited to 12 and 72 h. Nonablative Er‐YAG laser irradiation (SP Spectro, Fotona, Slovenia) was applied to the hESC culture within a biosafety cabinet with laminar airflow. Laser pulses were delivered consecutively and perpendicularly to the culture dish from a distance of 1 cm, with four shots per point, using a 7 mm spot size. The non‐ablative Er‐YAG laser with 2940 nm wavelength operated at 1.4 Hz and 1.5 J/cm 2 , in SMOOTH mode, following the protocol of Gaspar et al. [ 17 ] To evaluate the remodeling effect of Er‐YAG laser on endometrial tissue, matrix metalloproteinase‐2 (MMP‐2) levels in conditioned media were measured by an enzyme‐linked immunosorbent assay (ELISA) (Elabscience; E‐EL‐H1445, Houston, TX, USA), which has a sensitivity of 0.47 ng/mL and no reported cross‐reactivity or interference [ 18 ]. To assess whether the Er‐YAG laser induces T helper type 1 (Th1)‐ and Th2‐like effects on hESCs, we measured tumor necrosis factor‐alpha (TNF‐α) cytokine [ 19 ] and interleukin‐6 (IL‐6) cytokines [ 20 ] levels in conditioned media using ELISA kits (Elabscience; E‐EL‐H0109, E‐EL‐H6156, Houston, TX, USA). The kits had sensitivities of 4.69 pg/mL for TNF‐α and 0.94 pg/mL for IL‐6, with no reported cross‐reactivity or interference. To evaluate whether the Er‐YAG laser enhances angiogenesis in hESCs, vascular endothelial growth factor‐A (VEGF‐A) levels [ 21 ] in conditioned media were measured by ELISA (Elabscience; E‐EL‐H0111, Houston, TX, USA) with a sensitivity of 18.75 pg/mL and no reported cross‐reactivity or interference. To investigate the effect of the Er‐YAG laser on endometrial decidualization, we measured insulin‐like growth factor‐binding protein‐1 (IGFBP‐1) levels in conditioned media using ELISA kits (Elabscience; E‐EL‐H0442, Houston, TX, USA) [ 22 ] with a sensitivity of 0.10 ng/mL and no reported cross‐reactivity or interference. All ELISA analysis yielded R 2 values of 0.98–0.99, indicating good standard curves. MMP‐2, TNF‐α, IL‐6, VEGF‐A, and IGFBP‐1 levels were normalized to total cell culture protein, as measured by the bicinchoninic acid assay (BCA). After the experimental periods, conditioned media were collected, centrifuged at 1000 g for 20 min to remove cell debris, and the resulting supernatants were stored at −80°C. Samples were then added to 96‐well ELISA microplates coated with a capture antibody for 1.5 h, followed by the addition of a biotin‐conjugated detection antibody. Antibody binding was assessed using horseradish peroxidase‐conjugated avidin and a soluble colorimetric substrate, with absorbance measured at 450 nm using a microplate reader (Allsheng AMR‐100 microplate reader AS‐16050‐00). The main outcome of this study is to assess changes in MMP‐2, TNF‐α, IL‐6, VEGF‐A, and IGFBP‐1 levels in human ESC culture media following Er‐YAG laser irradiation at 12, 24, 48, and 72 h. SPSS version 26.0 (SPSS Science, Chicago, IL) was used for the statistical analysis of the data. The distribution of continuous data was assessed using the Shapiro–Wilk test. Non‐normally distributed data were presented as the median, 1st quartile (Q1) and 3rd quartile (Q3). The Mann–Whitney U test was used to compare independent, non‐normally distributed two variables, while the Kruskal–Wallis test was utilized for indepedent, non‐normally distributed more than two variables. Statistical significance was defined as p  < 0.05.