Association Between Pregnancy Outcomes and the Time of Progesterone Exposure of D6 Single-Blastocyst Transfer in Frozen-Thawed Cycles: A Retrospective Cohort Study.

Purpose: The objective of this study was to assess reproductive outcomes of D6 blastocysts transferred on day 6 in comparison to those transferred on day 7 of progesterone exposure in frozen-thawed embryo transfer cycles. Patients and Methods: This retrospective cohort study included 2029 D6 single blastocysts from the first frozen-thawed embryo transfer cycles of patients at the Hospital for Reproductive Medicine Affiliated to Shandong University from February 2017 to January 2020. Participants were divided into Group A (blastocyst transferred on the 6th day of progesterone exposure, n=1634) and Group B (blastocyst transferred on the 7th day of progesterone exposure, n=395). Results: The live birth rate was comparable between Group A and Group B (38.7% versus 38.7%, P=0.999). Subgroup analysis revealed a significantly higher preterm birth rate in D6 single blastocysts transferred on the 7th day than in those transferred on the 6th day of progesterone exposure for natural cycle frozen-thawed embryo transfer (5.2% versus 11.3%, P=0.020). After adjustment for potential confounders, the differences in the preterm birth rate in natural cycles persisted (adjusted odds ratio 2.347, 95% confidence interval 1.129-4.877, P=0.022). Conclusion: In frozen-thawed embryo transfer cycles, transferring on the 6th or 7th day of progesterone exposure of D6 blastocysts did not affect the live birth rate; however, when a natural cycle protocol is adopted, the possible preterm risk of transferring D6 blastocysts on the 7th day of progesterone exposure should be noted.

Highly expressed lncRNA H19 in endometriosis promotes aerobic glycolysis and Histone lactylation.

Previous studies from our group and others have shown increased IncRNA H19 expression in both the eutopic endometrium and the ectopic endometriosis tissue during endometriosis. In this study, we use immunofluorescence, immunohistochemistry and protein quantification to determine that levels of aerobic glycolysis and histone lactylation; which we show are increased in endometriosis tissues. In HESC cells (Human Endometrial Stromal Cells), we found that high H19 expression resulted in abnormal glucose metabolism by examining the levels of glucose, lactate, and ATP and measuring protein levels of enzymes that participate in glycolysis. At the same time, immunofluorescence and western blotting demonstrated increased histone lactylation in H19 overexpressing cells. Altering aerobic glycolysis and histone lactylation levels through the addition of Nala (sodium lactate) and 2-DG demonstrated that increased aerobic glycolysis and histone lactylation levels resulted in enhanced cell proliferation and cell migration, contributing to endometriosis. To validate these findings in vivo, we constructed an endometriosis mouse model, demonstrating similar changes in endometriosis tissues in vivo. Both aerobic glycolysis and histone lactylation levels were elevated in endometriotic lesions. Taken together, these data demonstrate elevated expression levels of H19 in endometriosis patients promote abnormal glucose metabolism and elevated histone lactylation levels in vivo, enhancing cell proliferation and migration and promoting the progression of endometriosis. Our study provides a functional link between H19 expression and histone lactylation and glucose metabolism in endometriosis, providing new insights into disease mechanisms that could result in novel therapeutic approaches.

Reproductive outcomes of single frozen-thawed embryo transfer in patients with endometriosis after preimplantation genetic testing.

PURPOSE: The reproductive outcomes of patients with endometriosis who are infertile have attracted recent attention. We aimed to explore whether endometriosis affects endometrial receptivity by observing pregnancy outcomes following a euploid blastocyst frozen embryo transfer. METHODS: This retrospective cohort study analyzed the data of patients with endometriosis from the reproductive hospital affiliated to Shandong University between January 2015 and December 2021. Control groups were matched using the 1:3 propensity score. The live birth, clinical pregnancy, biochemical pregnancy, clinical abortion, premature birth, and aneuploid rates were compared between the control group and endometriosis group. RESULTS: A total of 625 patients who underwent preimplantation genetic testing (PGT) prior to embryo implantation were included in the analysis. There were no significant differences in the live birth, clinical pregnancy, biochemical pregnancy, clinical abortion, and premature birth rates between the two groups. The aneuploidy rate of blastocysts obtained from the endometriosis group was higher than that of the control group (P = 0.012). CONCLUSION: Pregnancy outcomes using frozen embryos after PGT in patients with endometriosis did not differ from those in other women experiencing infertility. However, endometriosis may affect the quality of oocytes, resulting in a higher rate of aneuploidy.

Efficacy of atosiban for repeated implantation failure in frozen embryo transfer cycles.

Atosiban was commonly added to improve pregnancy outcomes of patients with repeated embryo implantation failure (RIF). In this study, we aimed to investigate the effect of atosiban before transferring the frozen-thawed embryo to RIF patients. This retrospective study was conducted in the Hospital for Reproductive Medicine affiliated to Shandong University from August 2017 to June 2021. A total of 1774 women with a history of RIF undergoing frozen embryo transfer (FET) were included in this study. All the participants were classified into atosiban or control group: Group A included 677 patients who were administered atosiban intravenously 30 min prior to FET with a dose of 37.5 mg; Group B included 1097 patients who received no atosiban before the transfer. There were no significant differences observed in the live birth rate (LBR) (39.73% vs. 39.02%, P = 0.928) between the two groups. Other secondary outcomes including biochemical pregnancy rate, clinical pregnancy rate, implantation rate, clinical miscarriage rate and preterm birth rate were similar between the two groups (all P > 0.05). However, subgroup analysis demonstrated significantly higher preterm birth rates in the control group compared with the atosiban group (0 versus 3.0%, P = 0.024) in the natural FET cycles. Atosiban may not improve pregnancy outcomes of RIF patients in FET cycles. However, the effects of Atosiban on pregnancy outcomes should be assessed in clinical trials with larger sample sizes.

6-(7-Nitro-2,1,3-benzoxadiazol-4-ylthio) Hexanol Inhibits Proliferation and Induces Apoptosis of Endometriosis by Regulating Glutathione S-Transferase Mu Class 4.

Endometriosis is a chronic disease associated with a disrupted oxidative balance and chronic inflammation. In this study, we investigated the role of glutathione S-transferase Mu class 4 (GSTM4) in endometriosis and determined whether 6-(7-nitro-2,1,3-benzoxadiazol-4-ylthio) hexanol (NBDHEX) regulates GSTM4 expression to affect cellular functions and oxidative stress. GSTM4 expression was detected by immunohistochemistry in endometrium from 15 endometriosis patients and 15 healthy controls. Western blotting was used to detect the expression of GSTM4, proliferating cell nuclear antigen (PCNA), matrix metalloproteinase-9 (MMP-9), Survivin, B-cell lymphoma-extra-large (Bcl-XL), Bax, kelch-like ECH-associated protein 1 (Keap1), and nuclear factor-erythroid 2-related factor 2 (Nrf2) in primary endometrial stromal cells with endometriosis (EESC) and normal endometrial stromal cells (NESC). The effects of NBDHEX on cell proliferation, migration, and invasion were evaluated using Cell Counting Kit-8 (CCK8) and Transwell assays. Apoptosis was detected by flow cytometry. The expression of GSTM4 was significantly increased in endometrium from endometriosis patients. Upon NBDHEX treatment, ESC exhibited reduced proliferation, migration and invasion abilities, and increased apoptosis. NBDHEX decreased the expression of endometriosis prognostic markers (PCNA and MMP-9) and anti-apoptotic proteins (Survivin and Bcl-xl), while it increased the expression of the apoptotic protein Bax. It had no effect on Keap1 expression, and it decreased the expression of Nrf2. The effect of siRNA-mediated knockdown of GSTM4 was similar to that of suppressing GSTM4 expression with NBDHEX treatment. These results indicate that GSTM4 is highly expressed in endometriosis and its expression is inhibited by NBDHEX. Decreased expression of GSTM4 inhibits cell growth, migration, and invasion, and negatively regulates Nrf2 to affect oxidative stress-induced apoptosis. Our results suggest that GSTM4 may play a role in ameliorating the progression of endometriosis. NBDHEX may have therapeutic potential in the treatment of endometriosis.

The Impact of Endometrioma on Embryo Quality in In Vitro Fertilization: A Retrospective Cohort Study.

The influence of endometrioma on oocyte and embryo competence is inconclusive. Furthermore, the benefits of surgical treatment remain uncertain. This study aimed to investigate the effect of endometrioma on oocyte and embryo quality from a morphological perspective and further explore whether surgery could contribute to improving oocyte and embryo competence. A total of 664 IVF cycles with endometrioma (538 cycles underwent surgeries) and 3133 IVF cycles from the control group were included. The propensity score matching was used to balance the baseline differences between groups. There was a lower MII oocyte rate (85.0% versus 87.8%, p < 0.001; 84.9% versus 87.6%, p = 0.001) and a similar good-quality embryos rate in women with endometrioma (and those who underwent surgeries) compared with control group. For women with endometrioma, the rates of blastocyst development (67.1% versus 60.2%; p = 0.013) and good blastocyst development (40.7% versus 35.2%; p = 0.049) were significantly higher in those who had undergone surgical treatment compared with those who had not, but the rates of MII oocytes (79.9% versus 87.7%; p < 0.001) and normal fertilization (55.2% versus 66.2%; p < 0.001) were lower. The study indicates that endometrioma, including its surgical treatment, compromises the oocyte maturity not the embryo quality at the cleavage stage; however, the surgery seems to contribute to improving blastocyst development.

The Effect of Short-Term Aspirin Administration during Programmed Frozen-Thawed Embryo Transfer on Pregnancy Outcomes and Complications.

Low-dose aspirin is widely used during frozen-embryo transfer (FET) cycles. Its anti-platelet property makes it a potentially useful drug for the prevention of hypertension disorders of pregnancy (HDP). However, the existing evidence about the effect of short-term aspirin administration on pregnancy outcomes is not clear. In our study, we retrospectively investigated women who had their first or second FET cycles at the Reproductive Hospital Affiliated with Shandong University from April 2017 to December 2020. A total of 4454 programmed FET cycles were recruited. According to whether aspirin was administrated in the protocols, the patients were divided into two groups: The Control group (n = 2793, 85 of them using donor sperm) and the Aspirin group (n = 1661, 35 of them using donor sperm). We analyzed the pregnancy outcomes and pregnancy complications of these cycles and observed similar live birth rates. We found that the short-term use of aspirin at a dosage of 50 mg per day for women undergoing programmed FET did not elevate the live birth rate or decrease the incidence of a series of pregnancy complications, including HDP. Based on our experience, short-term administration of low-dose aspirin may not improve the outcomes of young women undergoing frozen-thawed embryo transfer cycles.

Efficacy of long-term pituitary down-regulation pretreatment prior to in vitro fertilization in infertile patients with endometriosis: A meta-analysis.

Controversial conclusions have been made in previous studies regarding the influence of ultra-long gonadotropin-releasing hormone agonist (GnRH-a) in the reproductive outcomes of women with endometriosis who are undergoing in vitro fertilization/ intracytoplasmic sperm injection embryo transfer (IVF/ICSI-ET). An electronic search was conducted through PubMed, Embase, Cochrane Library, Web of Science, Elsevier ScienceDirect and Medline from inception until 10 September 2022. Only randomized studies were included. After the selection process, seven articles were eventually included in the meta-analysis. The pooling of the results showed the adverse effect of ultra-long protocol in terms of live birth rate (risk ratio (RR) = 0.53, 95% confidence intervals (CI): 0.31-0.9, P=0.02) and fertilization rate (RR = 1.18, 95% CI: 1.02-1.36, P=0.02). There was no statistical significance between the ultra-long protocol and long protocol of the rest outcome Indicators. The findings of this meta-analysis suggest that ultra-long GnRH-a does not appear to improve the results of IVF/ICSI treatment outcomes in patients with endometriosis.

Association of the Cumulative Live Birth Rate with the Factors in Assisted Reproductive Technology: A Retrospective Study of 16,583 Women.

The cumulative live birth rate (CLBR) can better reflect the overall treatment effect by successive treatments, and continuous rather than categorical variables as exposure variables can increase the statistical power in detecting the potential correlation. Therefore, the dose-response relationships might find an optimal dose for the better CLBR, offering evidence-based references for clinicians. To determine the dose-response relationships of the factors and the optimal ranges of the factors in assisted reproductive technology (ART) associated with a higher CLBR, this study retrospectively analyzed 16,583 patients undergoing the first in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI) from January 2017 to January 2019. Our study demonstrated the optimal ranges of age with a higher CLBR were under 32.10 years. We estimated the CLBR tends to increase with increased levels of AMH at AMH levels below 1.482 ng/mL, and the CLBR reaches a slightly high level at AMH levels in the range from 2.58-4.18 ng/mL. The optimal ranges of basal FSH with a higher CLBR were less than 9.13 IU. When the number of cryopreserved embryos was above 1.055 and the number of total transferred embryos was 2, the CLBR was significantly higher. In conclusion, there is a non-linear dose-response relationship between the CLBR with age, AMH, basal FSH, and the number of cryopreserved embryos and total transferred embryos. We proposed the optimal ranges of the five factors that were correlated with a higher CLBR in the first oocyte retrieval cycle, which may help consultation at IVF clinics.

Effect of different antibiotic therapies on the reproductive outcomes of fresh embryo transfer for chronic endometritis: A retrospective cohort study.

OBJECTIVE: To investigate the reproductive outcomes by comparing two kinds of antibiotic schemes for chronic endometritis (CE) in infertile women's fresh embryo transfer (FET) cycles and identify subgroups of patients with CE who need long-term antibiotics treatment. DESIGN: A retrospective cohort study. SETTING: University-based reproductive medical center. PATIENT(S): A total of 492 women with CD138-positive plasmacytes per 10 high-power fields (CD138+/10HPF). INTERVENTION(S): Hysteroscopy was performed and endometrial biopsy samples were collected in the proliferative phase. Long-term or short-term antibiotics were administrated. After antibiotics treatment, patients underwent in vitro fertilization (IVF)/ intracytoplasmic sperm injection (ICSI) and received ET. MAIN OUTCOME MEASURE(S): Ongoing pregnancy rate, clinical pregnancy rate, clinical miscarriage rate. RESULT (S): There were no significant differences in pregnancy outcomes between patients with CD138+/10HPF 1-4 (low-grade CE) who received long-term antibiotic therapy and short-term antibiotics groups. Among women with CD138+/10HPF ≥5 (high-grade CE), live birth rate (48.4% vs. 14.7%, p = .001), clinical pregnancy rate (66.7% vs. 35.3%, p = .002) and ongoing pregnancy rate (59.1% vs. 20.6%, p < .001) in the long-term arm were significantly higher than that in the short-term arm. The clinical miscarriage rate (21.0% vs. 58.3%, p = .013) was statistically lower in the long-term antibiotics group, but no statistical differences were found between the two groups in preterm delivery rate. CONCLUSION: Long-term antibiotics treatment was a sensible choice to improve pregnancy outcomes in women with CD138+/10HPF ≥5 (high-grade CE). The pregnancy outcomes of women with low-grade CE only defined by histological diagnosis were not greatly improved after antibiotic therapy. Therefore, we recommended the proper diagnosis criteria were CD138+/10HPF ≥5 pathologically.

Low STING expression promotes endometrial stromal cell invasion and migration via the STING/IRF-3/IFN-ß1 pathway in eutopic endometrium of women with endometriosis.

Aims: The primary aim of the current study was to elucidate the function of the stimulator of interferon genes (STING) in the eutopic endometrium of women with endometriosis. Materials and Methods: STING expression and signaling pathways were verified by western blot analysis and immunohistochemistry after si-STING treatment. Cell proliferation and invasion and migration were assessed using 5-ethynyl-2'-deoxyuridine and transwell assays, respectively. Results: Within endometriosis tissues, STING was primarily expressed in the stroma of the eutopic endometrium and glandular epithelium of the ectopic endometrium. However, STING expression was significantly lower in the eutopic endometrium of patients with endometriosis compared to controls (p < 0.05). Additionally, cell proliferation (0.2866 ± 0.01470 vs. 0.6911 ± 0.01796, ****p < 0.0001), invasion (130.0 ± 6.296 vs. 424.1 ± 22.31, ****p < 0.0001), and migration (82.93 ± 6.940 vs. 82.93 ± 6.940, ****p < 0.0001) were significantly increased in the si-STING groups. Moreover, following si-STING transfection, the expression of phosphorylated IRF-3 and TBK1 that are involved in STING/IRF3/IFNb1 signaling pathway decreased. The addition of exogenous IFN-ß1 effectively increased stromal cell invasion (IFN-ß1-NC vs. IFN-ß1-si-STING 274.7 ± 7.767 vs. 135.7 ± 12.63, ***p < 0.0001) and migration (IFN-ß1-NC and IFN-ß1-si-STING 28.53 ± 3.625 vs. 28.53 ± 3.625, ***p < 0.0001) without significantly impacting cell proliferation (si-STING vs. IFN-1ß-si-STING 0.6874 ± 0.02081 vs. 0.7187 ± 0.02638, p = 0.795). Conclusions: The STING signaling pathway plays an important role in endometrial stromal cell proliferation, invasion and migration associated with endometriosis.

The effect of endometriosis on IVF/ICSI and perinatal outcome: A systematic review and meta-analysis.

PURPOSE: The purpose of the study was whether endometriosis influences IVF/ICSI and subsequent pregnancy and obstetric outcomes. METHODS: Searches were performed on PubMed, EMBASE, Medline, and Web of Science. Most of the included studies were retrospective case control study and retrospective cohort study. The risk ratio with 95% confidence intervals were calculated using the random and fixed effects model. Random effects models were applied in the case of significant heterogeneity (I2>50%), and fixed effects models were used if the heterogeneity was not significant. The main outcome measure was the live birth rate. RESULTS: Seventy studies were included in our analysis. Compared with the control group, the implantation rate (p = 0.04) and the number of oocyte retrieved (p<0.00001) were significantly lower in women with endometriosis. About late pregnancy complications, there were significant statistical differences in multiple pregnancies rate, placenta previa rate, ectopic pregnancy rate, and postpartum hemorrhage rate between the two groups (p<0.05). All other major reproductive and obstetric outcomes were similar in women with and without endometriosis. CONCLUSIONS: Our results demonstrate women with or without endometriosis have similar reproductive outcomes, but women with endometriosis who conceived after assisted reproductive technology are in high risk pregnancy.

The newly non-uniform endometrial echogenicity on transvaginal ultrasound do not impact in vitro fertilization and embryo transfer success: A retrospective cohort study.

OBJECTIVE: To evaluate the effect of newly non-uniform endometrial echogenicity diagnosed by transvaginal ultrasound on the outcome of pregnancy in vitro fertilization-embryo transfer (IVF-ET) during controlled ovarian hyperstimulation (COH) by retrospective cohort analysis. METHODS: A retrospective cohort study of a total of 604 patients with newly non-uniform endometrial echogenicity from January 2013 to June 2017, each woman was matched with three control subjects of similar age (±1 year), type of infertility (primary or secondary), the protocol used for COH, and the number of ET cycles in our unit. The patients in the study group and control group were matched according to the strict 1:3 matching principle. Baseline characteristics and pregnancy outcomes were compared. RESULTS: There were no statistically significant difference in baseline characteristics, live birth rate, biochemical pregnancy rate, clinical pregnancy rate, clinical pregnancy miscarriage rate and ectopic pregnancy rate between the two groups. But there were significant statistical differences in past history of uterine cavity surgery between the two groups (35.26% VS 21.19%), especially in history of endometrial polyp surgery (94.84% VS 90.10%). CONCLUSIONS: Our results demonstrate the newly diagnosed non-uniform endometrial echogenicity during controlled ovarian hyperstimulation does not affect the pregnancy outcome of in vitro fertilization-embryo transfer, and fresh embryo transfer can be continued.

Effects of artificial cycles with and without gonadotropin-releasing hormone agonist pretreatment on frozen embryo transfer outcomes in patients with adenomyosis.

Gonadotropin-releasing hormone agonist (GnRH-a) is generally added to the improve pregnancy outcomes of hormone replacement therapy cycles among patients with adenomyosis. We aimed to investigate whether adding GnRH-a can result in better pregnancy outcomes. This retrospective analysis included 341 patients with adenomyosis who underwent frozen embryo transfer (FET) after in vitro fertilization (IVF). The control group was treated only with hormone replacement therapy cycles to prepare the endometrium, and GnRH-a was added to the study group before hormone administration to adjust the menstruation cycle. Based on the similar baseline values and embryological data, there was no significant difference in the clinical pregnancy rates (40.63% vs. 42.54%, P = 0.72) and live birth rates (23.75% vs. 23.75%, P = 0.74) of the control and study groups. Other secondary outcomes, including the rates of clinical miscarriage, ectopic pregnancy, preterm birth and term birth, were not significantly different between the two groups. Compared with the hormone replacement therapy cycle alone, GnRH-a downregulation based on a hormone replacement therapy cycle may not increase the rate of clinical pregnancy or live birth of IVF-ET with FET among infertile patients with adenomyosis.

Effect of Human Chorionic Gonadotropin Injection Before Frozen Embryo Transfer on Pregnancy Outcomes in Endometriosis-Associated Infertility.

Purpose: The aim of this study was to investigate the effect of human chorionic gonadotropin (hCG) in hormone replacement (HT) regime for frozen thawed embryo transfer in women with endometriosis (EM). Methods: We performed a retrospective, database-search, cohort study and included data on EM patients who underwent frozen embryo transfer (FET) between January 1, 2009 and August 31, 2018. According to the protocols for FET cycle, the patients were divided into two groups: control group (n = 296) and hCG group (n = 355). Clinical pregnancy rate, live birth rate, early abortion rate, late abortion rate, and ectopic pregnancy rate were compared between the two groups. Results: There was a significant increase in clinical pregnancy rate in the hCG group (57.7 vs. 49%, p = 0.027) compared with the control group. The live birth rate in the hCG group (45.6 vs. 38.5%, p = 0.080) was also elevated, but this difference was not statistically significant. Conclusion: hCG administration in HT regime for FET increases the pregnancy rate in women with EM.

Platelets impair natural killer cell reactivity and function in endometriosis through multiple mechanisms.

Study question: Do platelets have any role in the reduced cytotoxicity of natural killer (NK) cells in endometriosis? Summary answer: Platelets impair NK cell reactivity and function in endometriosis through multiple mechanisms. What is known already: Platelets play an important role in the development of endometriosis, and platelet-derived transforming growth factor-ß1 (TGF-ß1) suppresses the expression of NK Group 2, Member D (NKG2D) on NK cells, resulting in reduced cytotoxicity in women with endometriosis. Study design size, duration: Experiments on mice with induced endometriosis in which either platelets, NK cells or both were depleted and controls (none depleted). In vitro experiments with NK cells, platelets and, as target cells, endometriotic epithelial cell and endometrial stromal cell lines. Participants/materials setting methods: Immunohistochemistry analysis of ectopic endometrial tissues from mice with induced endometriosis receiving either platelet depletion (PD), NK cell depletion, or both or none. Immunofluorescence, flow cytometry and gene expression analysis for major histocompatibility complex class I (MHC-I) expression in target cells. Cytotoxicity and degranulation assays and the measurement of interferon (IFN)-γ secretion for the evaluation of NK cytotoxicity. Flow cytometry and gene expression for the expression of NK cell receptors. Main results and the role of chance: PD resulted in significantly reduced lesion weight in mice with induced endometriosis, but NK cell depletion as well as concomitant platelet and NK cell depletion increased the weight, suggesting that the anti-endometriosis effect of PD is mediated, at least in part, by increased NK cell cytotoxicity against endometriotic cells. Co-incubation of target cells with platelets resulted in rapid platelet coating as well as increased MHC-I expression in these cells, effectively providing a cloak of 'pseudo-self' to these cells to shield against NK cell lysis. It also reduced the expression of NKG2D ligands MICA and MICB and reduced the NK cell cytotoxicity. In addition, co-incubation of NK cells with platelets impaired the NK cell cytotoxicity as well. This impaired NK cell cytotoxicity was not due to the increased NK cell apoptosis, but, rather, through reduced NK cell degranulation and IFN-γ production, and reduced expression of activating receptors NKG2D and NKp46 and increased expression of inhibitory receptor KIR2DL1 in NK cells. Inhibition of TGF-ß1 signaling partially restored the aberrant expression of NKG2D, NKp46 and KIR2DL1, and partially restored the impaired NK cell cytotoxicity induced by activated platelets and their releasate. Large scale data: Not applicable. Limitations reasons for caution: This study is confined by the limitation of animal and in vitro experimentation and the lack of direct human data. Wider implications of the findings: Anti-platelet treatment holds promise in treating endometriosis. Study funding/competing interests: The National Natural Science Foundation of China (81471434 to S.W.G., 81270676 to S.W.G., 81370695 to X.S.L. and 81671436 to X.S.L). None of the authors has anything to disclose.

Platelet-derived TGF-ß1 mediates the down-modulation of NKG2D expression and may be responsible for impaired natural killer (NK) cytotoxicity in women with endometriosis.

STUDY QUESTION: Does platelet-derived transforming growth factor-ß1 (TGF-ß1) have any role in the reduced cytotoxicity of natural killer (NK) cells in women with endometriosis? SUMMARY ANSWER: Platelet-derived TGF-ß1 suppresses the expression of NK Group 2, Member D (NKG2D) on NK cells, resulting in reduced cytotoxicity in women with endometriosis, but neutralization of TGF-ß1 reverses the reduction. WHAT IS KNOWN ALREADY: NK cells are cytotoxic lymphocytes that play an important role in peritoneal immune surveillance, and their function is known to be impaired in women with endometriosis. There is increased platelet aggregation in endometriotic lesions and increased platelet activation rate in the peripheral blood in women with endometriosis, yet activated platelets release copiousTGF-ß1, which is known to be a potent immunosuppressive molecule that suppresses NK cell function and NKG2D expression. STUDY DESIGN, SIZE, DURATION: Cross-sectional clinical studies of 30 women with endometriosis and 33 women without endometriosis and in vitro experimentation with and without TGF-ß1 blockade. PARTICIPANTS/MATERIALS, SETTING, METHODS: Peritoneal fluid (PF) samples from premenopausal women with endometriosis and age- and menstrual phase-matched controls were collected. Platelet count, white blood cell (WBC) count, mean platelet volume (MPV), platelet activation rate, TGF-ß1 concentration, expression levels of NKG2D on NK cells in the PF were evaluated. The apoptosis of freshly isolated NK cells treated with PF from women with endometriosis, the NK cytotoxicity and NKG2D expression treated with PF in the presence or absence of an anti-TGF-ß1 antibody were also determined. MAIN RESULTS AND THE ROLE OF CHANCE: The platelet count, WBC count, MPV, platelet activation rate and the TGF-ß1 concentration in the PF from women with endometriosis were significantly elevated when compared with those of women without endometriosis. The TGF-ß1 concentration correlated positively with the platelet activation rate (r = 0.59, P < 0.01), suggesting that activated platelets are responsible, at least in part, for the increased TGF-ß1 concentration. The cytotoxicity of freshly isolated NK cells treated with PF of women with endometriosis is significantly reduced when compared with that of women without endometriosis. Both the platelet activation rate and the TGF-ß1 concentration in the PF correlated negatively with the NKG2D expression in NK cells isolated from the PF (r = -0.36, P < 0.01, and r = -0.45, P < 0.01, respectively). In addition, the NKG2D expression level and the cytotoxicity in freshly isolated NK cells were found to be significantly reduced if co-cultured with PF from women with endometriosis, but the TGF-ß1 blockade effectively reverses the reduction. LIMITATIONS, REASONS FOR CAUTION: This study is limited by the cross-sectional nature of the study. WIDER IMPLICATIONS OF THE FINDINGS: NKG2D is known to potently activate NK cells, so potent that it even overrides inhibitory signals transduced by other inhibitory receptors. This is the first time we demonstrate that platelet-derived TGF-ß1 may be responsible for reduced NKG2D expression as well as reduced cytotoxicity of NK cells in women with endometriosis. This study provides yet another piece of evidence that platelets play critical roles in the development of endometriosis, and anti-platelet treatment should improve NK cell functionality in treating endometriosis. Equally important, this study highlights the critical role of the lesion microenvironment in shaping NK cell-mediated anti-endometriotic immunity. STUDY FUNDING/COMPETING INTERESTS: This research was supported in part by grants 81270676 (S.-W.G.), 81471434 (S.-W.G.), 81530040 (S.-W.G.), and 81370695 (X.L.) from the National Natural Science Foundation of China, and grant 2013ZYJB0019 (X.L.) from Shanghai Municipal Commission of Health and Family Planning. None of the authors has anything to disclose.

Endometriosis-Derived Stromal Cells Secrete Thrombin and Thromboxane A2, Inducing Platelet Activation.

Platelets have been recently revealed to play important roles in the development of endometriosis. However, it is unclear whether endometriotic lesions can secrete any platelet inducers outside the menstruation window. Hence, this study was undertaken to see whether endometriosis-derived stromal cells secrete platelet activators and cause platelet activation. We employed in vitro experimentation using primary ectopic endometrial stromal cells (EESCs) and platelets from healthy male volunteers and evaluated the extent of platelet aggregation by aggregometer and the platelet activation rate by flow cytometry using supernatants harvested from EESCs of different cell densities. We also measured the concentration of thromboxane B2 (TXB2), a metabolite of thromboxane A2 (TXA2), and thrombin activity in supernatants harvested from EESCs of different densities and evaluated the extent of platelet aggregation after treatment of EESCs with hirudin, Ozagrel, and apyrase. Finally, the concentration of TXB2, thrombin, and transforming growth factor ß1 (TGF-ß1) in platelets cocultured with different densities of EESCs is measured by enzyme-linked immunosorbent assay. We found that EESCs secrete thrombin and TXA2 and induce platelet activation and aggregation in a density-dependent fashion. Treatment of platelets with EESCs resulted in increased concentration of TXB2, thrombin, and TGF-ß1 in a density-dependent manner. Treatment of EESCs with hirudin and Ozagrel, but not apyrase, resulted in significant suppression of platelet aggregation. Thus, given recently reported effects of activated platelets on the cell behaviors of EESCs and endometriotic lesions in general, our findings establish that endometriotic lesions and platelets engage active cross-talks in the development of endometriosis, highlighting the importance of lesion microenvironment in endometriosis.

Endocrine and inflammatory factors and endometriosis-associated infertility in assisted reproduction techniques.

PURPOSE: Our research aimed to evaluate the effect of endometriosis on folliculogenesis and pregnancy, and to assess the involvement of inflammatory factors (IL1b, PGE2, PGF2α, and TGFß2) in follicular fluid. METHODS: A total of 65 follicular fluid aspirates were collected. Concentrations of inflammatory factors (IL1b, PGE2, PGF2α, and TGFß2) and steroid hormones (E2, progesterone, FSH, and LH) within follicular fluid as well as serum E2 and LH concentrations were measured. The mRNA expression of IL1b, Ptgs2, aromatase, and PPARγ in granulosa cells was determined. The outcome of ART was monitored and recorded. RESULTS: The oocyte retrieval, rate of metaphase II oocyte, cleavage rate, effective embryo rate, and pregnancy rates of patients with endometriosis were all significantly lower than those of the control patients. In those with endometriosis, serum E2 concentrations were lower than those observed in controls. Aromatase levels in the granulosa cells of the endometriosis group were lower while concentrations of PGE2 in follicular fluid were higher than in the control group. Concentrations of PGE2, PGF2α, TGFß2, and IL1b were significantly correlated with each other. CONCLUSIONS: These results suggest that the outcomes of ART, in relation to serum E2 concentration, were adversely affected by the presence of endometriosis. Furthermore, the results supported that, among the endocrine and inflammatory factors, PGE2 within the follicular fluid impairs the number and quality of oocytes.