Abstract Study question Is it possible to assess the ultrasonographic attributes of preovulatory ovarian stigmas (PS) and the risks of follicle persistence in natural cycles in subfertile women? Summary answer TVUS using the Mobile Hydroacoustic Window (MHAW) technique reveals the subtle echostructure of preovulatory stigma (PS) and helps to assess the risk of follicle persistence. What is known already It is known that the preovulatory follicle takes an oval shape. The thinning of the follicle walls in the PS-area is described. Two variants of PS have also been shown to be associated with normal and aberrant ovulation as well as associated corpus luteum patterns. Ultrasound technique MHAW, which uses peritoneal fluid of biological origin for additional contrasting, made it possible to study some subtle objects of the pelvis, including ultrasound attributes of PS. Study design, size, duration Cross-sectional prospective observational study. The study was performed at the Remedi Clinic Odessa between April 2018 and January 2019. Informed consent was obtained from all study participants. Participants/materials, setting, methods 75 subfertile women were divided into two groups: group I (n = 59) with confirmed ovulation and group II (n = 16) with confirmed LUF (10) or HAF (6). All women underwent daily modified TVUS (MHAW) starting on day 9 of the natural cycle. Ultrasonographic attributes of PS were assessed during the last five days before ovulation (group I) or until the day of the first signs of follicular persistence (group II). Main results and the role of chance The oval pattern of the preovulatory follicle was registered in 83.1% of cases in group I and in 18.8% in group II. The mean volume of peritoneal effusion on the last day of PS assessment in group I was 2.9 versus 0.9 in group II. Thinning of the membranes of the ovaries/follicles, mainly in the PS area, was confirmed in 76% of all cases and in 96.6% in group I. The average wall thickness in day 0/-1 before ovulation, in group I was 0.5 mm versus 1.2 mm on the day the first manifistations of luteinization appeared, in group II. Wall thickness on the last day of measurement correlated with follicle size. PS dome formation was detected in 49.2% of ovulated women. Only in group I, in 39% of cases, in the last days before ovulation, the “Constriction ring phenomenon” was detected. Clinical variants of follicle persistence were confirmed, such as HAF 6 (8%) and LUF 10 (13.3%). A mean growing follicle size more than 28 mm is associated with an increased risk of follicle persistence. MHAW may be useful in determining the optimal timing of oocyte retrieval and studying the risks of follicle persistence in natural IVF/IUI cycles. Limitations, reasons for caution There are no limitations and reasons for caution Wider implications of the findings The problem of ultrasound diagnosis of subtle pelvic echostructures using MHAW-technique requires further extended research using control and in-depth statistical analysis Trial registration number 0117U007494 (Ukrainian state registration number)