Tubal occlusion in patients with hydrosalpinx modifies the uterine environment and improves in vitro fertilization outcomes within 7 months

Patients diagnosed with hydrosalpinx who visited the Reproductive Hospital of the Zhuang Autonomous Region between January 2016 and December 2022 for IVF or intracytoplasmic sperm injection (ICSI) were considered for inclusion. All patients underwent either proximal tubal occlusion or distal salpingostomy prior to controlled ovarian hyperstimulation (COH) and embryo transfer (ET). The exact day of oocyte retrieval after tubal occlusion was recorded, with 30 days defined as 1 month. The eligibility criteria were as follows: age of 20–45 years; regular menstruation; participation in a long gonadotropin-releasing hormone (GnRH) agonist protocol; hysterosalpingography (HSG) or vaginal ultrasound showing hydrosalpinx in one or both fallopian tubes, for which laparoscopic proximal occlusion and distal stoma of both fallopian tubes had been performed; IVF/ICSI performed within 365 days; and negative test results for chlamydia/gonorrhea infection. The exclusion criteria were endometriosis, uterine adenomyosis, prior ovarian surgery, uterine malformations (e.g., mediastinum, unicornis, or bicornis), endometrial lesions, severe intrauterine adhesions, other endocrine disorders, or impaired ovarian function due to radiotherapy, chemotherapy, or autoimmune disease. For patients with hydrosalpinx who first underwent occlusion and then IVF, fresh embryo transfer was carried out when conditions were suitable. If fresh transfer failed, frozen–thawed embryo transfer was performed. Single blastocyst transfer was generally preferred to maximize safety and outcomes. However, for older patients or those with prior failed implantation, two embryos could be considered. Regardless of whether cleavage-stage embryos or blastocysts were used, no more than two embryos were transferred. The primary endpoint was defined as either a live birth during the IVF cycle or exhaustion of all embryos available for transfer. All participants provided informed consent before laparoscopic bilateral proximal tubal occlusion. The study was approved by the Ethics Committee of the Reproductive Hospital of Guangxi Zhuang Autonomous Region in accordance with the Declaration of Helsinki (Approval No.: KY-LL-2022-13). Clinical information specific to this study was collected, including age; body mass index (BMI); antral follicle count (AFC); infertility duration; basal follicle-stimulating hormone (FSH), luteinizing hormone (LH), and estradiol (E2) levels; and the proportion of primary versus secondary infertility. Ovarian response was assessed by monitoring LH, progesterone (P), and E2 levels, along with other factors such as the initial gonadotropin (Gn) dose, total Gn dose for ovulation stimulation, duration of Gn administration (days), endometrial thickness on the human chorionic gonadotropin (hCG) trigger day, and the number of oocytes retrieved. Embryo and oocyte data were also recorded, including oocyte maturation rate, normal fertilization rate, total cleavage rate, number of available cleavage-stage (D3) embryos, good-quality D3 embryo rate, blastocyst (D5/D6) formation rate, and high-quality blastocyst formation rate. When one principal follicle reached ≥ 18 mm in diameter, or two principal follicles reached ≥ 17 mm, or three principal follicles reached ≥ 16 mm, and hormone levels were optimal, 6000–8000 IU urinary-derived hCG [recombinant 250 µg hCG (Merck Serono S.p.A) combined with 2000 IU hCG (Livzon Pharmaceutical Group Inc.)] was administered as a trigger for ovulation. Transvaginal ultrasound-guided oocyte retrieval was then performed 34–36 h after hCG administration. Embryos classified as Grade II with ≥ 6 cells and derived from 2PN were rated as high-quality D3 embryos, while Grade III embryos with ≥ 4 cells were rated as usable embryos. Blastocysts graded 3BB or above were defined as high-quality blastocysts, whereas those graded 3BC or above were considered usable blastocysts [ 16 ]. Subgroup analysis was based on the interval between tubal occlusion and oocyte retrieval, which was divided into 13 months using 30 days as 1 month. For further analyses, patients were grouped into ≤ 3 months, 3–7 months, and > 7 months. Patients aged ≥ 35 years were classified as advanced age, and low ovarian reserve was defined as an antral follicle count (AFC) of < 5–7 or anti-Müllerian hormone (AMH) level of < 1.1 ng/mL. Statistical analysis was performed according to the interval between tubal occlusion and oocyte retrieval. One-way analysis of variance or the Welch test was used for continuous variables, and the chi-square test was applied for categorical variables. Logistic regression analyses were conducted to evaluate the association between the timing of oocyte retrieval after tubal occlusion and pregnancy outcomes, including clinical pregnancy, abortion, multiple pregnancy, ectopic pregnancy, and live birth. Binary logistic regression was performed using unadjusted, age-adjusted, and multivariate-adjusted models. In the multivariate-adjusted models, the following covariates were included: age, BMI, AFC, infertility duration, basal FSH, basal LH, and basal E2 levels. Additionally, the time to oocyte retrieval after tubal occlusion was divided into three groups (≤ 3 months, 3–7 months, and > 7 months), with the ≤ 3 month group used as the reference to further assess the potential influence of occlusion timing on pregnancy outcomes. All analyses were carried out using SPSS version 24.0 (IBM Corp., Armon, NY, USA). All statistical tests were two-tailed, and P  < 0.05 was considered statistically significant. Curve estimation was conducted to identify the optimal timing of oocyte retrieval after tubal occlusion using binary logistic regression analysis. Dependent factors included clinical pregnancy, abortion, multiple pregnancy, ectopic pregnancy, and live birth, with adjustments made for age, BMI, AFC, infertility years, and basal FSH, LH, and E2 levels. The probability of pregnancy outcomes was applied in curve estimation using linear, logarithmic, inverse, quadratic, cubic, compound, power, S, growth, exponential, and logistic models. The R square value, adjusted R square value, and P -value were used to evaluate model fit, with the size of R square indicating the rationality of model construction. P  < 0.05 was considered statistically significant. Piecewise regression analysis was performed to identify the time point of oocyte retrieval after occlusion at which adverse assisted pregnancy outcomes occurred. In this method, the independent variables follow a linear relationship within a certain range, while another linear equation with a different slope is applied in other ranges. The segmented package in R software (version 4.4.1) was used. Logistic regression distribution probabilities of clinical pregnancy, live birth, and IVF-ET pregnancy time points were used to construct a linear regression model for estimating the segmented node. In addition, with clinical pregnancy outcome and live birth as dependent variables and the oocyte retrieval time after occlusion as the independent variable, generalized linear models were constructed for piecewise regression analysis, and the segmented node was again obtained. Mediation effect analysis was conducted to explore the role of ovarian reserve and hormone levels in influencing IVF-ET pregnancy outcomes because previous analyses had identified significant differences in these factors after tubal occlusion. The principle is as follows: when the independent variable X influences the dependent variable Y, and this influence occurs through another variable M, then M is considered a mediating variable. The effect of X on Y through M is referred to as the mediation effect (ME). In this causal pathway, M lies between X and Y, meaning that X leads to Y indirectly via M. Based on this framework, AFC, LH, E2, and FSH were used as mediating variables to compare the effects of IVF-assisted pregnancy timing (≤ 3 months, 3–7 months, and > 7 months after occlusion) on outcomes such as clinical pregnancy, live birth, miscarriage, and multiple pregnancy. The mediation package in R 4.4.1 was applied for the analysis. Both linear regression and generalized linear regression models were used, with multivariable adjustments for age, BMI, infertility duration, and infertility type. The final evaluation indices included average causal mediation effect (ACME, indirect effect), average direct effect (ADE), and total effect (TE), which together explain the association between X and Y through the mediating variables. Ovarian reserve and hormone levels were prospectively followed in patients scheduled for laparoscopic proximal fallopian tube disconnection combined with distal salpingostomy due to hydrosalpinx at Guangxi Reproductive Hospital between October 2021 and December 2023. Changes in ovarian function and hormone levels were recorded before occlusion and at 1, 2, 3, and 7 months after occlusion to evaluate the potential influence of ovarian reserve and hormone levels on assisted pregnancy outcomes. In total, 10 endometrial tissue samples were collected at the Reproductive Hospital of Guangxi Zhuang Autonomous Region between January 2024 and July 2024, including 5 samples obtained before tubal occlusion and 5 samples collected within 7 months after occlusion, under hysteroscopic guidance. Endometrial sampling was performed during the follicular phase, within 2–5 days after the end of menstruation. These samples were used to explore possible molecular mechanisms underlying the improved pregnancy outcomes observed within 7 months of tubal occlusion. All patients provided written informed consent prior to surgery. RNA was extracted using a commercial RNA extraction kit (Qiagen) according to the manufacturer’s instructions and subsequently used for RNAseq. Differential expression gene analysis of RNAseq was performed as follows. TrimGalore ( https://github.com/FelixKrueger/TrimGalore/ ) was first used for data quality control, with the Phred quality score threshold set to 20 and read length > 20, along with other default parameters. Alignment of clean reads was then carried out using HISAT2 (version 2.1.0) with the NCBI GRCh38 reference genome ( https://genome-idx.s3.amazonaws.com/hisat/grch38_genome.tar.gz ). Gene expression levels were quantified with FeatureCounts ( http://bioinf.wehi.edu.au/FeatureCounts/ ). Differentially expressed genes (DEGs) were identified using the DESeq2 package, comparing samples before and after occlusion. Visualization of results was performed with the ggplot and pheatmap packages. All analyses were conducted in R version 4.3.1. The clusterProfiler package was used for KEGG enrichment and GO annotation of DEGs. Significant genes ( P  1) were used to interpret the corresponding pathways and functional changes before and after tubal occlusion. GSEA ( http://software.broadinstitute.org/gsea/index.jsp ) was performed to identify pathway activation before and after occlusion using DEGs ( P  0.6). The Molecular Signatures Database (MSigDB, c2.cp.kegg.v7.4.symbols.gmt) was used as the reference set. KEGG signaling pathways were considered significantly enriched with P  2.0. CIBERSORTx was used to estimate the abundance of cell types within the mixed cell population. The input signature matrix file consisted of 22 immune cell gene expression profiles provided by CIBERSORTx, while the mixture file was the RNAseq expression matrix before and after tubal occlusion. Analysis was performed using 1000 permutation tests. Single-cell data ( GSE183837 ) from normal endometrium were obtained from the GEO database ( https://www.ncbi.nlm.nih.gov/geo/ ). The expression matrix was processed using the Seurat R package (version 4.0.3), with strict quality control to ensure reliable results. Genes expressed in fewer than 10 cells and cells expressing fewer than 200 genes were excluded. To avoid low-quality or double cells, only cells with fewer than twice the median number of genes were retained, and the proportion of mitochondrial genes was limited to < 40%. To minimize potential batch effects, the functions SelectIntegrationFeatures , FindIntegrationAnchors , and IntegrateData in Seurat were applied to merge the single-cell data. Based on ElbowPlot results, 20 principal components were selected for dimensionality reduction using uniform manifold approximation and projection. The resolution parameter was set to 0.2 to identify distinct cell clusters. DEGs were calculated using the likelihood ratio test implemented in FindAllMarkers and FindMarkers . Endometrial tissues were fixed in 4% paraformaldehyde for 24 h, then dehydrated and embedded in paraffin blocks. The blocks were sectioned into 4-µm pathological slices and stained using immunohistochemistry and immunofluorescence kits. For immunohistochemistry, the M2 macrophage marker CD163 (ab182422, diluted 1:500) was used. For immunofluorescence, primary antibodies included CD163 (ab182422, 1:500) and KRT8/18 (MA5-14088, 1:250). Alexa Fluor 488–conjugated donkey anti-mouse (1:200) and Alexa Fluor 555–conjugated donkey anti-rabbit (1:200) were used as secondary antibodies. Images were captured with an Olympus microscope.

In this study, 976 patients with hydrosalpinx were recruited. To identify the optimal timing of oocyte retrieval after occlusion, logistic regression analysis was performed. The results indicated that delaying oocyte retrieval after occlusion significantly reduced clinical pregnancy rates (unadjusted: odds ratio [OR] = 0.882, 95% confidence interval [CI] = 0.836–0.931, P  < 0.001; multivariate-adjusted: OR = 0.904, 95% CI = 0.851–0.959, P  = 0.001) and live birth rates (unadjusted: OR = 0.908, 95% CI = 0.863–0.956, P  < 0.001; multivariate-adjusted: OR = 0.926, 95% CI = 0.874–0.982, P  = 0.010) (Table  1 ). To further define the time points for oocyte retrieval, curve estimation was performed for the probability of clinical pregnancy (R 2  = 0.264) and live birth (R 2  = 0.184), with the cubic curve showing the best fit. Both curves suggested higher rates of clinical pregnancy and live birth within 3 months (Fig.  1 ). In addition, linear regression and piecewise logistic regression analyses indicated that 7 months might represent a critical time point, after which the probability of clinical pregnancy and live birth declined (Fig.  2 ). Table 1 Logistic regression analysis of pregnancy outcomes and IVF-ET assisted pregnancy interval after tubal occlusion Unadjusted Multi-adjusted OR 95%CI P OR 95%CI P Clinical pregnancy 0.882 0.836–0.931 < 0.001* 0.904 0.851–0.959 0.001* Abortion 0.982 0.897–1.075 0.696 1.035 0.932–1.149 0.519 Multiple pregnancies 1.021 0.963–1.083 0.484 1.045 0.981–1.112 0.175 Live birth 0.908 0.863–0.956 < 0.001* 0.926 0.874–0.982 0.010* * Multivariate correction factors included age, BMI, AFC, infertility years, basal FSH level, basal LH level, basal E 2 level, total number of embryos transferred, Initial dose of Gn, total Gn, number of eggs harvested and number of high quality blastocyst. * indicated a statistical difference Logistic regression analysis of pregnancy outcomes and IVF-ET assisted pregnancy interval after tubal occlusion * Multivariate correction factors included age, BMI, AFC, infertility years, basal FSH level, basal LH level, basal E 2 level, total number of embryos transferred, Initial dose of Gn, total Gn, number of eggs harvested and number of high quality blastocyst. * indicated a statistical difference Fig. 1 Curve estimation of clinical pregnancy ( a ) and live birth ( b ) probability from Logistic regression with the time of oocyte retrieval after tubal occlusion. * the left is all the curve modes, and the right is the most significant curve mode Curve estimation of clinical pregnancy ( a ) and live birth ( b ) probability from Logistic regression with the time of oocyte retrieval after tubal occlusion. * the left is all the curve modes, and the right is the most significant curve mode Fig. 2 Piecewise logistic regression predicted the time node of oocyte retrieval after tubal occlusion according to the clinical pregnancy and live birth rate. ( a ) Linear model; ( b ) Generalized Linear model Piecewise logistic regression predicted the time node of oocyte retrieval after tubal occlusion according to the clinical pregnancy and live birth rate. ( a ) Linear model; ( b ) Generalized Linear model According to the curve and piecewise analyses, 976 patients were divided into three groups (≤ 3 months, 3–7 months, and > 7 months after occlusion). Significant differences were observed in age (≤ 3 months: 33.11 ± 4.36; 3–7 months: 32.65 ± 4.33; >7 months: 34.18 ± 3.85; P  7 months: 4.36 ± 3.91; P  = 0.046), and total number of embryos transferred (≤ 3 months: 2.42 ± 1.34; 3–7 months: 2.69 ± 1.38; >7 months: 2.21 ± 1.17; P  7 months: 20.70 ± 9.44; P  = 0.017) and an increase in basal FSH levels (≤ 3 months: 7.32 ± 2.00; 3–7 months: 7.34 ± 1.70; >7 months: 7.76 ± 1.83; P  = 0.008) were observed with longer intervals before oocyte retrieval. The number of oocytes retrieved (≤ 3 months: 13.94 ± 6.91; 3–7 months: 13.68 ± 6.01; >7 months: 12.49 ± 6.90; P  = 0.048), available D3 embryos (≤ 3 months: 10.34 ± 5.40; 3–7 months: 10.24 ± 4.78; >7 months: 8.86 ± 5.69; P  = 0.001), and high-quality blastocyst formation rate (≤ 3 months: 39.75%; 3–7 months: 39.16%; >7 months: 35.14%; P  = 0.014) all declined with later oocyte retrieval. Conversely, the rate of biochemical abortion increased (≤ 3 months: 1.52%; 3–7 months: 5.24%; >7 months: 6.71%; P  = 0.011), while cumulative live birth rates decreased markedly (≤ 3 months: 76.34%; 3–7 months: 71.53%; >7 months: 58.77%; P   7 months ( N  = 228) P Age (years) 33.11 ± 4.36 32.65 ± 4.33 34.18 ± 3.85 < 0.001 Infertility age (years) 5.01 ± 3.50 5.10 ± 3.90 4.36 ± 3.91 0.046 BMI (kg/m 2 ) 21.75 ± 2.56 21.93 ± 2.88 22.15 ± 2.96 0.363 AFC (NO.) 23.03 ± 9.40 22.61 ± 8.85 20.70 ± 9.44 0.017 Basel FSH level (mIU/ml) 7.32 ± 2.00 7.34 ± 1.70 7.76 ± 1.83 0.008 Basel E₂ level (pg/ml) 46.07 ± 26.82 44.57 ± 27.86 42.86 ± 26.31 0.487 Basel LH level (mIU/ml) 5.40 ± 2.11 5.50 ± 2.39 5.51 ± 2.29 0.897 AMH (ng/ml) 3.06 ± 1.66 2.67 ± 1.40 2.53 ± 1.60 0.286 Embryo transfer conditions Fresh 116 (43.94%) 598 (39.60%) 192 (37.35%) Frozen 148 (56.06%) 912 (60.40%) 322 (62.65%) 0.206 Total number of embryos transferred 2.42 ± 1.34 2.69 ± 1.38 2.21 ± 1.17 < 0.001 Infertility type Primary infertility (%) 31.18(58/186) 33.63(189/562) 28.07(64/228) Secondary infertility (%) 68.82 (128/186) 66.37 (373/562) 71.93 (164/228) 0.308 Initial dose of Gn (IU) 202.55 ± 54.88 198.13 ± 56.53 216.78 ± 62.83 < 0.001 Total Gn (IU) 2434.95 ± 946.42 2345.73 ± 975.14 2610.96 ± 1062.81 0.003 Gn days 10.61 ± 1.64 10.74 ± 1.73 10.65 ± 1.74 0.593 endometrial thickness at the time of hCG injection (mm) 10.57 ± 1.92 10.71 ± 1.94 10.86 ± 1.83 0.311 E 2 level at the time of hCG injection (pg/ml) 2377.47 ± 987.59 2366.93 ± 1142.75 2246.53 ± 1201.18 0.353 LH level at the time of hCG injection (mIU/ml) 1.89 ± 1.33 2.41 ± 10.92 1.92 ± 1.04 0.649 Prog level at the time of hCG injection (ng/ml) 0.67 ± 0.31 0.70 ± 0.36 0.68 ± 0.34 0.494 Number of eggs harvested 13.94 ± 6.91 13.68 ± 6.01 12.49 ± 6.90 0.048 MII rate (%) 81.48 (2112/2592) 81.92 (6298/7688) 80.76 (2300/2848) 0.389 2PN rate (%) 73.20 (1546/2112) 71.39 (4496/6298) 69.91 (1608/2300) 0.054 Number of available D3 embryos 10.34 ± 5.40 10.24 ± 4.78 8.86 ± 5.69 0.001 High quality D3 embryo formation rate (%) 47.30 (910/1924) 47.01 (2706/5756) 46.04 (930/2020) 0.688 Available blastocyst formation rate (%) 62.80 (888/1414) 63.57 (2610/4106) 58.75 (846/1440) 0.005 High quality blastocyst formation rate (%) 39.75 (562/1414) 39.16 (1608/4106) 35.14 (506/1440) 0.014 Average frequency of embryos transferred 1.70 ± 0.35 1.72 ± 0.31 1.72 ± 0.36 0.212 Frequency of transplantation (time/period) 1.42 ± 0.66 1.56 ± 0.73 1.31 ± 0.64 < 0.001 Unusable embryo rate (%) 3.76(7/186) 1.07(6/562) 3.95(9/228) 0.014 Blood HCG positive rate (%) 62.88(166/264) 60.59 (532/878) 59.73 (178/298) 0.663 Biochemical abortion (%) 1.52(4/264) 5.24 (46/878) 6.71 (20/298) 0.011 Clinical pregnancy rate (%) 61.36(162/264) 54.90 (482/878) 53.02 (158/298) 0.104 Early abortion rate (%) 6.17(10/162) 11.20 (54/482) 12.66 (20/158) 0.118 Late abortion rate (%) 6.17(10/162) 5.39 (26/482) 2.53 (4/158) 0.265 Ectopic pregnancy rate (%) 0(0/264) 0.46 (4/878) 0 (0/298) 0.277 Multiple pregnancy rate (%) 25.93(42/162) 22.82 (110/482) 36.71 (58/158) 0.003 Live birth rate (%) 53.79(142/264) 45.79 (402/878) 44.97 (134/298) 0.053 Cumulative live birth rate (%) 76.34 (142/186) 71.53 (402/562) 58.77 (134/228) < 0.001 * Data given as mean ± SD, AFC: Antral follicle count, AMH: anti-mullerian hormone, BMI: Body mass index, FSH: Follicle-stimulating hormone, SD: Standard deviation, LH: Luteinizing hormone. Comparisons of basic information after tubal occlusion * Data given as mean ± SD, AFC: Antral follicle count, AMH: anti-mullerian hormone, BMI: Body mass index, FSH: Follicle-stimulating hormone, SD: Standard deviation, LH: Luteinizing hormone. Further logistic regression confirmed that oocyte retrieval > 7 months after occlusion significantly reduced both clinical pregnancy (unadjusted: OR = 0.447, 95% CI = 0.290–0.690, P  < 0.001; age-adjusted: OR = 0.478, 95% CI = 0.308–0.742, P  = 0.001; multivariate-adjusted: OR = 0.557, 95% CI = 0.344–0.901, P  = 0.017) and live birth rates (unadjusted: OR = 0.436, 95% CI = 0.285–0.666, P  < 0.001; age-adjusted: OR = 0.469, 95% CI = 0.305–0.724, P  = 0.001; multivariate-adjusted: OR = 0.524, 95% CI = 0.327–0.839, P  = 0.007), even after full adjustment (Table  3 ). Table 3 Logistic regression analysis of pregnancy outcomes for three groups Unadjusted Age-adjusted Multi-adjusted OR 95% CI P OR 95% CI P OR 95% CI P Clinical pregnancy ≤ 3 months Ref. Ref. Ref. Ref. Ref. Ref. Ref. Ref. Ref. 3–7 months 0.879 0.593–1.303 0.521 0.843 0.566–1.255 0.400 0.885 0.575–1.364 0.580 > 7 months 0.447 0.290–0.690 < 0.001 0.478 0.308–0.742 0.001 0.557 0.344–0.901 0.017 Early abortion ≤ 3 months Ref. Ref. Ref. Ref. Ref. Ref. Ref. Ref. Ref. 3–7 months 1.795 0.893–3.607 0.101 0.785 0.523–1.179 0.244 1.696 0.793–3.623 0.173 > 7 months 1.328 0.588–3.001 0.495 1.281 0.807–2.033 0.638 1.928 0.795–4.674 0.146 Multiple pregnancy ≤ 3 months Ref. Ref. Ref. Ref. Ref. Ref. Ref. Ref. Ref. 3–7 months 0.816 0.545–1.220 0.321 0.785 0.523–1.179 0.244 0.813 0.531–1.243 0.338 > 7 months 1.170 0.742–1.844 0.499 1.281 0.807–2.033 0.293 1.483 0.913–2.409 0.111 Live birth ≤ 3 months Ref. Ref. Ref. Ref. Ref. Ref. Ref. Ref. Ref. 3–7 months 0.721 0.494–1.051 0.089 0.676 0.459–0.995 0.047 0.714 0.470–1.084 0.114 > 7 months 0.436 0.285–0.666 < 0.001 0.469 0.305–0.724 0.001 0.524 0.327–0.839 0.007 * OR: odds ratios, 95% CI: confidence interval, Multiple logistic regression models adjusting for age, BMI, AFC, infertility years, basal FSH level, basal LH level, basal E 2 level, total number of embryos transferred, Initial dose of Gn, total Gn, number of eggs harvested and number of high quality blastocyst Logistic regression analysis of pregnancy outcomes for three groups * OR: odds ratios, 95% CI: confidence interval, Multiple logistic regression models adjusting for age, BMI, AFC, infertility years, basal FSH level, basal LH level, basal E 2 level, total number of embryos transferred, Initial dose of Gn, total Gn, number of eggs harvested and number of high quality blastocyst We further analyzed the influence of age on IVF-ET outcomes after occlusion. Consistent with the overall results, in younger women (< 35 years), delaying oocyte retrieval significantly reduced both clinical pregnancy (unadjusted: OR = 0.405, 95% CI = 0.212–0.775, P  = 0.006; age-adjusted: OR = 0.404, 95% CI = 0.211–0.772, P  = 0.006; multivariate-adjusted: OR = 0.387, 95% CI = 0.194–0.775, P  = 0.007) and live birth rates (unadjusted: OR = 0.389, 95% CI = 0.212–0.714, P  = 0.002; age-adjusted: OR = 0.387, 95% CI = 0.211–0.711, P  = 0.002; multivariate-adjusted: OR = 0.376, 95% CI = 0.195–0.724, P  = 0.003) at 3–7 months, with an even stronger effect observed beyond 7 months (Clinical pregnancy: unadjusted: OR = 0.246, 95% CI = 0.121–0.503, P  < 0.001; age-adjusted: OR = 0.250, 95% CI = 0.122–0.513, P  < 0.001; multivariate-adjusted: OR = 0.265, 95% CI = 0.123–0.572, P  = 0.001. Live birth: unadjusted: OR = 0.293, 95% CI = 0.148–0.579, P  < 0.001; age-adjusted: OR = 0.300, 95% CI = 0.151–0.595, P  = 0.001; multivariate-adjusted: OR = 0.317, 95% CI = 0.152–0.664, P  = 0.002). In addition, oocyte retrieval beyond 7 months increased the risk of multiple pregnancy in younger women (unadjusted: OR = 1.786, 95% CI = 0.985–3.239, P  = 0.056; age-adjusted: OR = 1.939, 95% CI = 1.062–3.541, P  = 0.031; multivariate-adjusted: OR = 2.476, 95% CI = 1.289–4.754, P  = 0.006). By contrast, in older women (≥ 35 years), retrieval at 3–7 months was associated with an increased chance of clinical pregnancy (multivariate-adjusted: OR = 2.178, 95% CI = 1.099–4.318, P  = 0.026) and a reduced risk of multiple pregnancy (multivariate-adjusted: OR = 0.411, 95% CI = 0.195–0.864, P  = 0.019) (Table  4 ). Table 4 Logistic regression analysis of pregnancy outcomes for young or elderly patients Unadjusted Age-adjusted Multi-adjusted OR 95% CI P OR 95% CI P OR 95% CI P < 35 years Clinical pregnancy ≤ 3 months Ref. Ref. Ref. Ref. Ref. Ref. Ref. Ref. Ref. 3–7 months 0.405 0.212–0.775 0.006 0.404 0.211–0.772 0.006 0.387 0.194–0.775 0.007 > 7 months 0.246 0.121–0.503 < 0.001 0.250 0.122–0.513  7 months 2.000 0.585–6.839 0.269 1.803 0.522–6.224 0.351 2.190 0.576–8.328 0.250 Multiple pregnancy ≤ 3 months Ref. Ref. Ref. Ref. Ref. Ref. Ref. Ref. Ref. 3–7 months 1.051 0.627–1.763 0.849 1.032 0.614–1.735 0.904 1.181 0.677–2.060 0.558 > 7 months 1.786 0.985–3.239 0.056 1.939 1.062–3.541 0.031 2.476 1.289–4.754 0.006 Live birth ≤ 3 months Ref. Ref. Ref. Ref. Ref. Ref. Ref. Ref. Ref. 3–7 months 0.389 0.212–0.714 0.002 0.387 0.211–0.711 0.002 0.376 0.195–0.724 0.003 > 7 months 0.293 0.148–0.579 < 0.001 0.300 0.151–0.595 0.001 0.317 0.152–0.664 0.002 ≥ 35 years Clinical pregnancy ≤ 3 months Ref. Ref. Ref. Ref. Ref. Ref. Ref. Ref. Ref. 3–7 months 1.727 0.995-3.000 0.052 1.601 0.911–2.814 0.102 2.178 1.099–4.318 0.026 > 7 months 0.787 0.434–1.425 0.429 0.728 0.396–1.340 0.728 1.290 0.612–2.718 0.503 Early abortion ≤ 3 months Ref. Ref. Ref. Ref. Ref. Ref. Ref. Ref. Ref. 3–7 months 1.868 0.745–4.686 0.183 1.967 0.779–4.966 0.152 2.155 0.761–6.102 0.148 > 7 months 0.872 0.290–2.624 0.807 0.898 0.298–2.712 0.849 1.640 0.455–5.917 0.450 Multiple pregnancy ≤ 3 months Ref. Ref. Ref. Ref. Ref. Ref. Ref. Ref. Ref. 3–7 months 0.513 0.266–0.989 0.046 0.470 0.241–0.915 0.026 0.411 0.195–0.864 0.019 > 7 months 0.676 0.330–1.387 0.286 0.633 0.306–1.311 0.218 0.754 0.328–1.733 0.506 Live birth ≤ 3 months Ref. Ref. Ref. Ref. Ref. Ref. Ref. Ref. Ref. 3–7 months 1.208 0.706–2.066 0.490 1.097 0.631–1.906 0.742 1.236 0.665–2.295 0.503 > 7 months 0.660 0.366–1.193 0.169 0.599 0.325–1.102 0.100 0.767 0.383–1.539 0.456 * OR: odds ratios, 95% CI: confidence interval, Multiple logistic regression models adjusting for age, BMI, AFC, infertility years, basal FSH level, basal LH level, basal E 2 level, total number of embryos transferred, Initial dose of Gn, total Gn, number of eggs harvested and number of high quality blastocyst Logistic regression analysis of pregnancy outcomes for young or elderly patients * OR: odds ratios, 95% CI: confidence interval, Multiple logistic regression models adjusting for age, BMI, AFC, infertility years, basal FSH level, basal LH level, basal E 2 level, total number of embryos transferred, Initial dose of Gn, total Gn, number of eggs harvested and number of high quality blastocyst Patients were divided into normal and low ovarian reserve groups. In women with normal ovarian reserve, delaying oocyte retrieval after occlusion—particularly beyond 7 months—significantly reduced the likelihood of clinical pregnancy (unadjusted: OR = 0.456, 95% CI = 0.288–0.721, P  = 0.001; age-adjusted: OR = 0.485, 95% CI = 0.305–0.772, P  = 0.002; multivariate-adjusted: OR = 0.572, 95% CI = 0.344–0.954, P  = 0.032) and live birth (unadjusted: OR = 0.439, 95% CI = 0.281–0.686, P  < 0.001; age-adjusted: OR = 0.473, 95% CI = 0.300–0.746, P  = 0.001; multivariate-adjusted: OR = 0.546, 95% CI = 0.322–0.899, P  = 0.017). By contrast, in women with low ovarian reserve, the timing of oocyte retrieval showed no significant influence on pregnancy outcomes (Table  5 ). Table 5 Logistic regression analysis of pregnancy outcomes for patients with different ovarian reserve Unadjusted Age-adjusted Multi-adjusted OR 95% CI P OR 95% CI P OR 95% CI P Normal ovarian reserve Clinical pregnancy ≤ 3 months Ref. Ref. Ref. Ref. Ref. Ref. Ref. Ref. Ref. 3–7 months 0.931 0.616–1.409 0.736 0.888 0.584–1.349 0.557 0.966 0.614–1.518 0.879 > 7 months 0.456 0.288–0.721 0.001 0.485 0.305–0.772 0.002 0.572 0.344–0.954 0.032 Early abortion ≤ 3 months Ref. Ref. Ref. Ref. Ref. Ref. Ref. Ref. Ref. 3–7 months 1.630 0.805-3.300 0.175 1.771 0.868–3.610 0.116 1.652 0.758–3.599 0.207 > 7 months 1.425 0.629–3.229 0.396 1.295 0.568–2.953 0.538 2.035 0.827–5.003 0.122 Multiple pregnancy ≤ 3 months Ref. Ref. Ref. Ref. Ref. Ref. Ref. Ref. Ref. 3–7 months 0.837 0.554–1.266 0.400 0.799 0.526–1.214 0.293 0.817 0.528–1.265 0.365 > 7 months 1.176 0.732–1.890 0.502 1.286 0.795–2.080 0.306 1.414 0.853–2.341 0.179 Live birth ≤ 3 months Ref. Ref. Ref. Ref. Ref. Ref. Ref. Ref. Ref. 3–7 months 0.763 0.514–1.133 0.180 0.709 0.474–1.061 0.095 0.772 0.499–1.194 0.244 > 7 months 0.439 0.281–0.686 < 0.001 0.473 0.300-0.746 0.001 0.546 0.332–0.899 0.017 Low ovarian reserve Clinical pregnancy ≤ 3 months Ref. Ref. Ref. Ref. Ref. Ref. Ref. Ref. Ref. 3–7 months 0.556 0.149–2.072 0.381 0.683 0.174–2.674 0.584 1.186 0.169–8.317 0.864 > 7 months 0.476 0.119–1.902 0.294 0.444 0.107–1.850 0.265 0.393 0.052–2.994 0.367 Early abortion ≤ 3 months Ref. Ref. Ref. Ref. Ref. Ref. Ref. Ref. Ref. 3–7 months NA NA NA NA NA NA NA NA NA > 7 months NA NA NA NA NA NA NA NA NA Multiple pregnancy ≤ 3 months Ref. Ref. Ref. Ref. Ref. Ref. Ref. Ref. Ref. 3–7 months 0.537 0.087–3.322 0.503 0.565 0.090–3.560 0.543 0.905 0.050-16.449 0.946 > 7 months 1.435 0.248–8.291 0.687 1.430 0.247–8.277 0.690 5.136 0.375–70.448 0.221 Live birth ≤ 3 months Ref. Ref. Ref. Ref. Ref. Ref. Ref. Ref. Ref. 3–7 months 0.425 0.114–1.583 0.202 0.514 0.131–2.010 0.339 0.794 0.113–5.580 0.816 > 7 months 0.476 0.119–1.902 0.294 0.441 0.106–1.846 0.263 0.391 0.054–2.857 0.355 * OR: odds ratios, 95% CI: confidence interval, Multiple logistic regression models adjusting for age, BMI, AFC, infertility years, basal FSH level, basal LH level, basal E 2 level, total number of embryos transferred, Initial dose of Gn, total Gn, number of eggs harvested and number of high quality blastocyst Logistic regression analysis of pregnancy outcomes for patients with different ovarian reserve * OR: odds ratios, 95% CI: confidence interval, Multiple logistic regression models adjusting for age, BMI, AFC, infertility years, basal FSH level, basal LH level, basal E 2 level, total number of embryos transferred, Initial dose of Gn, total Gn, number of eggs harvested and number of high quality blastocyst Overall, the results identified the negative impact of delayed oocyte retrieval on pregnancy outcomes after occlusion. Both FSH levels and AFC varied significantly across different retrieval intervals, suggesting a potential role of hormones and ovarian reserve in influencing outcomes. Mediation effect analysis was then performed comparing retrieval times (≤ 3 months vs. 3–7 months; ≤3 months vs. >7 months). In this analysis, the ADE confirmed an association between retrieval time and pregnancy outcomes. However, hormones and ovarian reserve showed no significant ACME, indicating no mediating effect of these factors on the relationship between retrieval time and outcomes (Fig.  3 ). Fig. 3 Mediation effect analysis of ovarian reserve and hormone levels for the pregnancy outcomes in different time after tubal occlusion. ( a ) AFC; ( b ) E2; ( c ) FSH; ( d ) LH Mediation effect analysis of ovarian reserve and hormone levels for the pregnancy outcomes in different time after tubal occlusion. ( a ) AFC; ( b ) E2; ( c ) FSH; ( d ) LH To further validate this, 117 new patients were prospectively recruited (Table S1 ). In this cohort, an increase in FSH levels and a decrease in AFC were observed within 3 months, but both indices gradually returned to preoperative levels after 3 months. Similar patterns were noted across different ages and ovarian reserve groups (Table S2 ). These temporary changes further explain the absence of mediation effects of hormones and ovarian reserve on pregnancy outcomes. To investigate potential changes in the endometrial microenvironment before and after occlusion in patients with hydrosalpinx, bulk RNAseq was performed on endometrial samples. Differential expression analysis identified 203 genes (e.g., LINC01391 , GNLY , ITGAD , IL2RB ) and 407 genes (e.g., CXCL14 , CLIC6 , FCGBP , PLAG1 , ITM2A ) significantly expressed before and after occlusion, respectively (Fig.  4 b). GO annotation revealed prominent immune-related molecular functions before occlusion, such as MHC class Ib receptor activity (Fig.  4 c). KEGG pathway analysis also suggested activation of immune signaling prior to occlusion, including human T-cell leukemia virus 1 infection, natural killer cell–mediated cytotoxicity, cellular senescence, antigen processing and presentation, and complement and coagulation cascades. By contrast, after occlusion, cAMP signaling and cGMP-PKG signaling pathways were activated (Fig.  4 d). GSEA further confirmed enrichment of immune-related pathways in the endometrium before occlusion, compared with the postoperative state (Fig.  4 e). Fig. 4 RNAseq of the endometrium before and after tubal occlusion of hydrosalpinx. ( a ) differentially expressed genes (DEGs) of RNAseq; ( b ) the GO annotations of DEGs before and after occlusion; ( c ) KEGG enrichment; ( d ) GSEA analysis of pathways RNAseq of the endometrium before and after tubal occlusion of hydrosalpinx. ( a ) differentially expressed genes (DEGs) of RNAseq; ( b ) the GO annotations of DEGs before and after occlusion; ( c ) KEGG enrichment; ( d ) GSEA analysis of pathways The immune cell composition of the endometrium before and after occlusion was analyzed, revealing notable changes in the immune microenvironment (Fig.  5 a). T follicular helper cells were more abundant in the endometrium of patients with hydrosalpinx ( P  = 0.02), whereas M2 macrophage infiltration increased after occlusion ( P  = 0.029) (Fig.  5 b). Top DEGs identified after occlusion, such as CXCL14 , were also significantly correlated with M2 macrophages (Fig.  5 c). Furthermore, immunohistochemistry confirmed greater infiltration of M2 macrophages in the endometrium following occlusion (Fig.  5 d). Fig. 5 M2 macrophage polarization after tubal occlusion of hydrosalpinx in the endometrium. ( a ) immune cell components in the endometrium before and after tubal occlusion; ( b ) the proportion of T follicular helper and M2 macrophage; ( c ) correlations of Top DEGs before and after occlusion with T and M2 macrophage; ( d ) CD163 expression in the endometrium before and after tubal occlusion identified with immunohistochemistry M2 macrophage polarization after tubal occlusion of hydrosalpinx in the endometrium. ( a ) immune cell components in the endometrium before and after tubal occlusion; ( b ) the proportion of T follicular helper and M2 macrophage; ( c ) correlations of Top DEGs before and after occlusion with T and M2 macrophage; ( d ) CD163 expression in the endometrium before and after tubal occlusion identified with immunohistochemistry After occlusion of hydrosalpinx, pregnancy outcomes improved. To explore the formation of an immunosuppressive microenvironment in the endometrium, single-cell RNAseq data from normal endometrium were analyzed, identifying nine distinct cell types (Fig.  6 a). CXCL14 , the most highly expressed gene after occlusion, was localized primarily to epithelial cells of the endometrium, a finding further validated by immunofluorescence (Fig.  6 b). Although no specific receptor for CXCL14 has been identified, it has been reported to bind with high affinity to CXCR4 . In the single-cell data, CXCR4 expression was detected in macrophages, suggesting that CXCL14 may act through CXCR4 on macrophages to promote M2 macrophage polarization (Fig.  6 c). Fig. 6 Single-cell RNAseq of endometrium identified highly expressed CXCL14 gene in the epithelium. ( a ) single-cell RNAseq defined 9 cell types; ( b ) the CXCL14 and CXCR4 expressed in the endometrium; ( c ) located the CXCL14 in the endometrial epithelium cells with immunofluorescence Single-cell RNAseq of endometrium identified highly expressed CXCL14 gene in the epithelium. ( a ) single-cell RNAseq defined 9 cell types; ( b ) the CXCL14 and CXCR4 expressed in the endometrium; ( c ) located the CXCL14 in the endometrial epithelium cells with immunofluorescence

Although hydrosalpinx is a common factor that can affect female infertility and significantly influence assisted reproduction technology (ART) outcomes, the mechanisms behind these effects remain unclear [ 2 ]. Hydrosalpinx is associated with a range of conditions, including pelvic inflammation, endometriosis, appendicitis, or previous pelvic and abdominal surgery [ 17 ]. Treatment of hydrosalpinx with salpingostomy, salpingectomy or tubal occlusion has been linked to beneficial improvements in pregnancy outcomes [ 18 ]. In 2017, Xu et al. [ 19 ] compared the effects of various methods of hydrosalpinx treatment before IVF, and found that laparoscopic salpingectomy and proximal tubal occlusion had similar positive effects on pregnancy outcomes in women with hydrosalpinx undergoing IVF. In support of this, Bi et al. [ 20 ] also reported that prior treatment of hydrosalpinx by salpingectomy and proximal tubal occlusion was superior to other surgical procedures with respect to IVF pregnancy outcomes. Similarly, in 2023, Yilei et al. [ 13 ] reported higher clinical pregnancy rates when hydrosalpinx was treated by salpingectomy before IVF. At the same time, oocyte retrieval conducted 4–6 and 7–12 months after salpingectomy was associated with even higher cumulative pregnancy and live birth rates. Although our previous analyses support the effectiveness of tubal occlusion before IVF for hydrosalpinx treatment, no exact oocyte retrieval time was defined [ 12 ]. Based on the present study, and analysis of logistic regression and mediation effects, we recommend that oocyte retrieval should be performed within 7 months after tubal occlusion for hydrosalpinx treatment, with an optimal oocyte retrieval time of 3 months. In addition, it should also be mentioned that younger female patients with normal ovarian reserves are significantly more likely to benefit from tubal occlusion for hydrosalpinx treatment before IVF. If other critical factors that influence fertility are suboptimal, simply treating the hydrosalpinx will not likely improve IVF pregnancy outcomes. In particular, age has been identified as a determining factor that strongly influences female reproductive function; with the prevalence of infertility directly correlating with increasing age [ 21 ]. In addition to hydrosalpinx, other complex factors such as hyperprolactinemia, pelvic adhesions, endometriosis, and lower ovulatory dysfunction, etc., can also have additive or synergistic effects on reducing female fertility [ 22 ]. Similarly, ovarian reserves are another crucial factor directly affecting pregnancy outcomes. Low ovarian reserves have been linked to reduced pregnant rates and poorer ART prognosis [ 23 , 24 ]. Tubal occlusion for the treatment of hydrosalpinx cannot reverse ovarian reserve function. Even in the early stages of tubal occlusion, ovarian reserves could already be suboptimal. Thus, our results also highlight the importance of careful pre-selection of patients for tubal occlusion in the context of IVF-ET treatment programs. Based on our results we recommend an optimal oocyte retrieval time of ≤ 7 months after tubal occlusion; and speculate that hydrosalpinx recurrence and endometrial deterioration could negatively affect IVF outcomes for oocytes retrieved later than 7 months after tubal occlusion. As shown in another study, hydrosalpinx was associated with a high recurrence rate; and it is plausible that hydrosalpinx recurrence rates may increase in correlation with post-operation time. Liu et al. reported treatment of 562 hydrosalpinx cases in patients between January 1, 2012 and December 31, 2016. Of these, hydrosalpinx was found to have reoccurred in 146 patients after treatment [ 25 ]. As shown for other surgical hydrosalpinx treatment methods, such as hydrosalpinx aspiration with or without sclerotherapy, overall hydrosalpinx recurrence rates can reach as high as 30% [ 1 ]. Hydrosalpinx recurrence can cause toxic effects that directly reduce sperm motility, embryo development and endometrial receptivity [ 9 ]. Although hormone levels and ovarian reserves fluctuated after tubal occlusion, they returned to pre-operation levels after 3 months. However, our analysis did not identify any potential mediator effects by hormone and ovarian reserve levels on the relationship between pregnancy outcomes and tubal occlusion. Thus, the common influences of hydrosalpinx, or hydrosalpinx recurrence, may be related to deterioration of the endometrial environment and detrimental molecular changes in the endometrium. Studies have shown that hydrosalpinx can interfere with embryo implantation in the endometrium [ 26 ]. In addition, changes in the expression levels of specific candidate genes and proteins in hydrosalpinx patients can also affect the embryo implantation process [ 27 ]. While hydrosalpinx is a result of pelvic inflammation, local inflammation of the uterus may also occur. In 2022, Zou et al. reported that both bilateral and simple hydrosalpinx were closely related to chronic endometritis [ 28 ]. Using electron microscopy, Ajonuma et al. [ 29 ] found serious loss of cilia and microvilli on the epithelial surface of the fallopian tube in the hydrosalpinx. Moreover, inflammatory cells could also be seen in the hydrosalpinx, in areas with flattened or no epithelial cells in the lumen, and in dilated blood vessels and/or lymphatic vessels, which also could have adverse effects on the reproductive process and IVF outcomes [ 29 ]. Our previous results show that IVF outcomes in patients with hydrosalpinx are significantly improved after tubal occlusion. Further analysis of gene expression changes in the endometrial tissues of patients before and after hydrosalpinx, indicate that immuno-inflammatory pathways in the endometrial are significantly activated before tubal occlusion: including of T cell, antigen presenting and natural killer cell signaling pathways. These results strongly suggest a relationship between activated inflammatory responses in the endometrial tissues of hydrosalpinx patients and the high failure rate of IVF-ET before occlusion treatment. In addition, we also detected activated cAMP signaling pathways in the endometrium after occlusion, which could effectively inhibit tubal fluid stimulation by isoproterenol [ 30 ]. Thus, inactivation of cAMP signaling during hydrosalpinx recurrence may also aggravate tubal fluid and the negative effects of effusion on IVF. Due to the activation of a large number of immune signaling pathways in the endometrium by hydrosalpinx, further analysis indicates that M2 macrophage levels increase after occlusion, resulting in inhibition of the local immune inflammatory response in the surrounding tissues. As an important component of the immune system, macrophages are thought to be important factors in female infertility. Studies have shown that macrophages can accumulate in large numbers in patients with ectopic tubal pregnancy, salpingitis, hydrosalpinx and endometriosis [ 31 ]. In addition, in a mycoplasma-induced hydrosalpinx model, activation of macrophages and pro-inflammatory pathways was shown to promote the persistence of hydrosalpinx [ 32 ]. Polarization of M2 macrophages in the endometrial may effectively reduce endometrial inflammation and the risk of infertility [ 33 ]. Combining single-cell and total RNA sequencing analyses, we found high expression of the CXCL14 gene in the endometrium after tubal occlusion, which is thought to be closely related to M2 macrophages polarization. In 2020, Lv et al. found in both in vivo and in vitro experiments that CXCL14 effectively inhibits polarization of M1-type macrophages, while promoting production of M2-type macrophages [ 34 ]. In 2023, He et al. also found in mouse models that CXCL14 effectively induces polarization of M2-type macrophages, thereby improving atherosclerosis [ 35 ]. At the same time, in tumors, increased CXCL14 also promoted polarization of M2-type macrophages [ 36 , 37 ]. In the present study, CXCL14 was mainly found to be highly expressed in the epithelial cells of normal endometrial tissues, which was also verified by immunofluorescence. Although the receptor for CXCL14 is not known at present, some studies have reported that the epithelial chemokine CXCL14 acts synergistically with CXCL12 through allosteric regulation of CXCR4 [ 38 ]. Interestingly, our data also indicate that CXCR4 is highly expressed in the macrophages of normal endometrial, which suggest that CXCL14 in the epithelial cells of the endometrium after occlusion might interact with CXCR4 in macrophages, helping to maintain a low inflammatory microenvironment that could ensure better IVF pregnancy outcomes.

Hydrosalpinx is a tubal factor strongly associated with female infertility and a high inflammatory microenvironment in the endometrium. Evidence suggests that tubal occlusion before IVF can result in improved IVF pregnancy outcomes. Increased CXCL14 gene expression in epithelial cells of the endometrium after tubal occlusion may promote polarization of M2-type macrophages. After tubal occlusion, we recommended that IVF should be conducted within 7 months for optimal pregnancy outcomes.

Female infertility is a serious problem with far-reaching consequences for humanity. Approximately 25% of female infertility cases have been attributed to tubal factors [ 1 ]; and 30% of these tubal factors are classified as hydrosalpinx [ 1 ], which may be associated with pelvic inflammation. Hydrosalpinx has been linked to a dramatic decrease in pregnancy rates following in vitro fertilization-embryo transfer (IVF-ET) [ 2 ], possibly due the presence of inflammation in the endometrium [ 3 ]. It is now widely accepted that hydrosalpinx significantly reduces pregnancy rates for patients undergoing assisted reproductive technology (ART) treatment. For example, the success rate of IVF-assisted pregnancy for patients with hydrosalpinx was found to be approximately 30–50% lower than for those without hydrosalpinx [ 4 ]. In 2019, Harb et al. [ 5 ] conducted a large-sample meta-analysis of 14 studies to explore the impact of hydrosalpinx on pregnancy outcomes. The results of the meta-analysis indicate that hydrosalpinx is associated with a significant increase in miscarriage rate. Moreover, successful hydrosalpinx treatment led to significant improvements in IVF-ET pregnancy outcomes, coupled with decreased miscarriage rates [ 5 ]. Moreover, Peng et al. found that hydrosalpinx could result in a marked increase in the occurrence of chronic endometritis [ 6 ]; while promoting infiltration of inflammatory cells in the endometrium and impairing reproductive outcomes [ 7 ]. Thus, successful treatment of hydrosalpinx may effectively prevent excessive inflammation in the endometrium, and have positive effects on IVF-ET outcomes. To date, several surgical procedures, including ultrasound-guided aspiration, laparoscopic salpingectomy, and proximal tubal occlusion + distal salpingostomy (hereafter tubal occlusion) have been successful in improving pregnancy outcomes when conducted before IVF-ET [ 8 ]. Ultrasound-guided aspiration of hydrosalpinx is a simple procedure that is associated with less damage and relatively lower cost than other methods. However, increased hydrosalpinx recurrence rates and lower efficacy with respect to improving pregnancy outcomes has restricted its application [ 9 , 10 ]. Before the advent of IVF, laparoscopic salpingectomy was a commonly used pre-treatment method for hydrosalpinx patients. However, the secondary damage to surrounding tissues associated with this surgical procedure could markedly reduce ovarian reserves and ovulation induction [ 9 , 10 ]. Laparoscopic bilateral tubal occlusion is a comparatively less invasive surgical procedure which could prevent the retrograde flow of hydrosalpinx fluid into the uterine cavity, improving endometrial receptivity, and achieving the same effect as fallopian tube resection. A 2020 meta-analysis reported that tubal occlusion could also significantly improve pregnancy outcomes compared with salpingectomy [ 11 ]. Although, prior surgical intervention was beneficial, the exact timing of oocyte retrieval following surgery was not reported [ 12 ]. Yilei et al. [ 13 ] recommended that oocyte retrieval should be performed within 4–12 months after salpingectomy, in order to improve pregnancy outcomes and live birth rates. As with other routine operations, tubal occlusion has also proved to be beneficial prior to IVF-ET, similar to other methods applied before IVF [ 14 ]. Moreover, tubal occlusion may result in significantly less damage to the ovarian reserve compared to salpingectomy [ 15 ]. To date, few studies have reported oocyte retrieval times following tubal occlusion, or its impact on the endometrial microenvironment.

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