Objective To investigate the factors influencing the characteristics of amniotic fluid sediment following second-trimester amniocentesis for prenatal diagnosis， and to evaluate their impact on amniotic fluid cell culture and pregnancy outcomes， thereby providing a basis for clinical specimen processing， risk assessment， and genetic counseling. Methods A total of 2 489 pregnant women who underwent mid-trimester （18 - 24 weeks） amniocentesis for singleton pregnancies at the Prenatal Diagnosis Center of the First Affiliated Hospital of Anhui Medical University from January 2025 to December 2025 were retrospectively reviewed. Among them， 96 cases with distinct sediment characteristics were ultimately included. Based on the visual characteristics and volume of the sediment from 20 mL centrifuged amniotic fluid aliquots， patients were categorized into four groups： small-volume milky-white sediment （Group A， n = 20）， large-volume milky-white sediment （Group B， n = 24）， small-volume bloody sediment （Group C， n = 29）， and small-volume dark-brown sediment （Group D， n = 23）. Baseline demographics and laboratory parameters were collected. Using the in situ amniotic fluid cell culture method， we recorded the culture duration， number of media changes， and total cell yield. Pregnancy outcomes were subsequently tracked. Statistical analyses were performed using one-way analysis of variance， chi-square tests， and binary logistic regression. Results There were statistically significant differences between the four groups in terms of RBC， CRP， culture cycle， number of fluid changes， cell count， and history of bleeding during pregnancy （all P < 0.05）. Among them， RBC and CRP levels were significantly different among the four groups. Group D had the highest RBC level， which was （4.93 ± 1.55）× 10¹²/L， which was significantly higher than other groups （F = 5.20， P = 0.002）； group B had the highest CRP level， which was （9.63 ± 2.87） mg/L， and the difference between the four groups was statistically significant （F = 5.69， P = 0.001）. In terms of culture-related indicators， there were significant differences among the four groups in terms of culture cycle， number of medium changes， and cell count （all P < 0.001）. Group D had the longest culture period， （13.52 ± 0.88） days； the most number of medium changes， （5.57 ± 0.650） times； and the lowest cell count， （1.09 ± 0.56）× 10⁵/mL. There were no statistically significant differences in conception methods， history of anticoagulant use， twin reduction， and placental position among the four groups （all P > 0.05）. The distribution of history of bleeding during pregnancy was significantly different among the four groups （χ2 = 14.04， P = 0.003）. Binary logistic regression model analysis showed that RBC， culture cycle， number of fluid changes， history of bleeding during pregnancy were significantly related to the occurrence of group D （OR = 2.27， 6.89， 4.25， 4.88 respectively）. Pregnancy outcomes showed that adverse events such as miscarriage， amniotic fluid leakage， and premature birth occurred in group D， while no adverse events occurred in the other three groups. Conclusions Amniotic fluid sediment characteristics exhibit significant variations during the second trimester. Specifically， dark-brown sediment is associated with impaired in vitro cell culture efficiency and an increased risk of adverse pregnancy outcomes. These findings underscore the necessity for early risk assessment， stratified specimen processing， and individualized prenatal management in clinical practice.