BackgroundOvarian tissue cryopreservation is a technique for fertility preservation addressed to prepubertal girls or to patients for whom no ovarian stimulation is possible before initiation of gonadotoxic treatments. Autotransplantation of frozen-thawed ovarian tissue is the only available option for reuse but presents some limitations: ischemic tissue damages post-transplant and reintroduction of malignant cells in cases of cancer. It is therefore essential to qualify ovarian tissue before autograft on a functional and oncological point of view. Here, we aimed to isolate viable cells from human ovarian cortex in order to obtain an ovarian cell suspension analyzable by multicolor flow cytometry.MethodsOvarian tissue (fresh or frozen-thawed), from patients with polycystic ovarian syndrome (reference tissue) and from patients who underwent ovarian tissue cryopreservation, was used for dissociation with an automated device. Ovarian tissue-dissociated cells were analyzed by multicolor flow cytometry; the cell dissociation yield and viability were assessed. Two automated dissociation protocols (named laboratory and commercial protocols) were compared.ResultsThe effectiveness of the dissociation was not significantly different between reference ovarian tissue (1.58 × 106 ± 0.94 × 106 viable ovarian cells per 100 mg of ovarian cortex, n = 60) and tissue from ovarian tissue cryopreservation (1.70 × 106 ± 1.35 × 106 viable ovarian cells, n = 18). However, the viability was slightly different for fresh ovarian cortex compared to frozen-thawed ovarian cortex whether we used reference tissue (p = 0.022) or tissue from ovarian cryopreservation (p = 0.018). Comparing laboratory and commercial protocols, it appeared that cell yield was similar but cell viability was significantly improved when using the commercial protocol (81.3% ± 12.3% vs 23.9% ± 12.5%).ConclusionBoth dissociation protocols allow us to isolate more than one million viable cells per 100 mg of ovarian cortex, but the viability is higher when using the commercial dissociation kit. Ovarian cortex dissociation is a promising tool for human ovarian cell qualification and for ovarian residual disease detection by multicolor flow cytometry.

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Study question What cancelation policy in controlled ovarian stimulation-intrauterine insemination (COS-IUI) cycles allows to lower the multiple pregnancy rate (MPR) without decreasing the live birth rate (LBR)? Summary answer An algorithm based on the woman’s age, serum Estradiol level and number of follicles ≥14 mm on trigger day reduces the MPR without impacting LBR. What is known already While the MPR in IVF cycles has significantly decreased in the past decades, it has remained stable and relatively high in COS-IUI cycles, at around 10-15%. The main reason behind this continuously high MPR in COS-IUI cycles has been the relative inability to lower it without significantly decreasing the overall pregnancy rates. Several risk factors are associated with MP in COS-IUI cycles, and recommendations have varied between the different scientific societies, but to date, there is no consensus on the best strategy to decrease the risk of MP in COS-IUI cycles without compromising the pregnancy and live birth rates. Study design, size, duration A bicentric observational cohort study at the Angers University Hospital (group A) and the Besançon University Hospital (group B) between January 2011 to December 2019. Approximately 350-400 IUI cycles are performed yearly in each center. All patients who had a clinical pregnancy following COS-IUI during the study period were included. Our main outcome measure was the MPR and our secondary outcome measures were the clinical pregnancy (CP), miscarriage and LBR. Participants/materials, setting, methods In group A, the starting gonadotropin dose was 50-100 IU/day, and the algorithm for cycle cancelation was based on the woman’s age, serum Estradiol (E2) level, and number of follicles ≥14 mm on trigger day. In group B, the starting gonadotropin dose was 100-150 IU/day and the cancelation policy was case-by-case and physician dependent, based on the woman’s age, number of follicles ≥15 mm, and number of previous failed COS-IUI cycles, without predefined cut-offs. Main results and the role of chance We included 6582 COS-IUI cycles (3387 in group A and 3195 in group B) that resulted in 884 clinical pregnancies (790 singletons, 86 twins and 8 triplets). The MPR was significantly lower in group A compared to group B (8.1% vs 13.3%, p = 0.01). The CPR (13.4% vs 13.4%, p = 0.99), the miscarriage rate (14.5% vs 15.6%, p = 0.64) and the LBR (10.8% vs 11.9%, p = 0.16) were comparable between groups A and B. Univariate analysis showed the following factors to be predictive of the risk of MP: the treatment center (OR = 1.73 [1.12-2.68]), the number of follicles ≥10 mm (OR = 1.22 [1.11-1.36]) and ≥14 mm on trigger day (OR = 1.43[1.20-1.70]). Multivariate analysis also showed the following factors to be predictive of the MP risk: the treatment center (aOR=1.63 [1.02-2.60]), the number of follicles ≥ 10 mm (aOR=1.20 [1.07-1.34]) and ≥14 mm on trigger day (aOR=1.39 [1.16-1.66]). The cycle cancelation rate was comparable between groups A and B (7.2% vs 7.2%, p = 0.93), while cycle cancelation rate for excessive response to COS was significantly lower in group A compared to group B (19.3% vs 35.9%, p < 0.001). The rate of divergence from cancelation protocol was significantly lower in group A compared to group B (0.09% vs 1.1% p < 0.001) Limitations, reasons for caution The main limitation of our study is the retrospective design. The algorithm needs to be tested in other populations for further validation. Wider implications of the findings The use of low starting doses of gonadotropins (50-100 IU/day), and the application of a strict algorithm that takes into account the woman’s age, serum E2 level and number of follicles ≥14 mm on trigger day allows to optimize the success rates of COS-IUI cycles Trial registration number Not applicable