Study question Can we use MPA as a pituitary inhibitor instead of the GnRH antagonist in ovarian stimulation protocols in non-oncological fertility preservation and PGT-A cycles? Summary answer MPA can act as a substitute of GnRH antagonist for pituitary suppression in FP and PGT-A cycles, since the results are similar between both groups. What is known already Progestin-primed ovarian stimulation (PPOS) protocols using exogenous progesterone to replace GnRH analogs during the follicular phase of OS have emerged as an efficient alternative to prevent LH from peaking and have been used successfully in different types of patients. Fertility preservation (FP) and preimplantation genetic testing (PGT-A) have become new emerging areas of assisted reproduction. FP gives women the ability to have children using their own gametes after age-related fertility decline, while PGT-A appears to improve reproductive outcomes in advanced maternal age at increased risk of aneuploid embryos. However, few data are available for both indications regarding PPOS cycle outcomes. Study design, size, duration Multicenter, retrospective, observational, cohort study conducted in eleven IVIRMA centers attached to private universities. We included a total of 4,961 cycles of non-oncological fertility preservation that were distributed as follows: n = 494 were stimulated under a PPOS protocol while n = 4,467 received a GnRH antagonist. Regarding PGT-A cycles, we analyzed 12,461 treatments, of which n = 686 and n = 11,775 received MPA and GnRH antagonist, respectively. Cycles were performed from January 2017 to December 2021. Participants/materials, setting, methods Patients were divided according to the protocol used for preventing premature luteinization during follicular phase of OS. In the MPA group, participants received 10 mg daily administered orally, while in the control group, women received an antagonist once the main follicle reached 13 mm. In FP cycles, ovarian response specific parameters were evaluated, such as endocrine profile and mature oocytes; in PGT-A treatments, main variables were number of biopsied and aneuploid embryos and reproductive outcomes. Main results and the role of chance Regarding FP's baseline characteristics, age was statistically but not clinically significant between the two groups. Length of ovarian stimulation and total dose of hMG administered were similar in both groups, despite the significantly higher total dose of FSH administered in MPA compared to the GnRH antagonist group (p = 0.008) . Number of mature oocytes retrieved (10.2 [95% CI 9.6-10.8] vs 9 [95% CI 8.8-9.2]) was significantly higher in MPA compared to antagonist group; this trend continued regardless of age (≤ 35 or > 35 years). PGT-A cycles followed the same tendency in terms of demographic characteristics. Length of OS was comparable between groups, whilst the total dose of rFSH and hp-HMG administered in the MPA were significantly higher than that in the GnRH antagonist group. Although the number of MII was comparable and despite the lower number of embryos biopsied in the MPA group (4.5±0.2 vs 4.7±0.06, p = 0.031) the number of aneuploid embryos was similar between the two groups (2.3±0.1 vs 2.4±0.04, p = 0.474), as well as implantation (56% vs. 54% p = 0.359) and clinical pregnancy rate (64.1% vs. 62.1, p = 0.316). The miscarriage rate was significantly lower in the group treated with MPA compared to GnRH antagonists (4.7% vs. 8.2%, p = 0.001). Limitations, reasons for caution The retrospective nature of this study may be a reason for caution and only association, not causation, can be inferred from the results. Despite being the largest sample size ever reported with PPOS in no oncological FP and PGT-A, the number of patients included is still low. Wider implications of the findings The administration of PPOS yielded similar or even better results than those observed with GnRH antagonists in terms of oocytes retrieved, rate of aneuploid embryos or clinical results. Therefore, PPOS could be recommended for ovarian stimulation in non-oncological FP and PGT-A cycles as it allows for a more patient-friendly approach. Trial registration number Not applicable

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Study question How powerful is the combination of artificial neural networks that combine embryo's protein profile with its automatic score provided by time-lapse videos to predict ploidy? Summary answer An artificial neural network (ANN) that considers proteomics and automatic score assigned by deep learning achieves 71% accuracy in distinguishing between euploid and aneuploid embryos. What is known already Currently the most widely used technique for detecting chromosomal abnormalities involves biopsy of the developing embryo. However, it has several disadvantages related to invasiveness, technical difficulty, high economic costs, etc. Therefore, different non-invasive techniques are being studied for the detection of aneuploid embryos. The discovery of cell-free DNA (cfDNA) released by the embryo to the culture media during its development marked the beginning of a new era of noninvasive PGT (niPGT) but some factors require adaptation for the analysis. Also, artificial Intelligence (AI) represents a valuable alternative to developing new models for predicting PGT outcomes without disturbing the embryo. Study design, size, duration This study included 294 samples of culture medium from 81 treatments of the PGT-A program. Out of the total, 23 were control samples (medium in which no embryos had been cultured) and 271 were samples where there was a developing embryo. Embryos were cultured until the blastocyst stage in EmbryoScope systems (Vitrolife, Sweden) with single-step medium (Gems, Genea) and automatically scored by iDAScore v2 algorithm from 1 to 9.9. Participants/materials, setting, methods The spent culture medium was collected on day 5/6 of embryo development and chromosome analysis was performed using next-generation sequence technology (Juno Genetics, Valencia). The relative concentrations of 92 proteins were analysed using Proseek Multiplex Assays (Olink Bioscience) using 1 μl of each sample. The final assay readout was presented as Normalized Protein eXpression (NPX) values. Finally, we developed our own ANN algorithms considering protein profile and automatic embryo score. Main results and the role of chance For euploid embryos (n = 101), 35 protein samples analysed had different NPX values between conditioned and control media*. The relative concentration was reduced for 11 proteins (consumption by the embryo) and 24 proteins increased their levels (secretion). For aneuploid embryos (n = 170), 33 protein samples analysed had different NPX values between conditioned and control media*. The relative concentration was reduced for 4 proteins and 29 proteins increased their levels. Out of the total, only 6 proteins had on average different concentrations between normal and abnormal embryos*: MCP_1, IL_17A, CXCL1, IL18, IL_22RA1 and CSF_1. Additionally, the automatic embryo score provided by iDAScore v2 algorithm was higher for euploid embryos than for aneuploid embryos (5.9 ± 2.7 vs. 5.1  ± 2.6)*. For ploidy prediction, three architectures of ANN were developed with different input data ANN1 (six discriminatory proteins), ANN2 (automatic embryo score) and ANN3 (six discriminatory proteins and automatic embryo score). Our dataset was divided into 68% training, 16% validation and 16% test. The accuracy, sensibility and specificity for the test phase were as follows: 63.6%, 76.9% and 44% for ANN1; 61%, 11.8% and 95.8% for ANN2; and 71.1%, 62.5% and 77.3% for ANN3. Limitations, reasons for caution Only one laboratory by using single step culture medium from one brand was involved in this study. Wider implications of the findings Our study showed a new approach to avoid transferring aneuploid embryos in cases where embryo biopsy is not performed. In addition, further studies on this field may result in a new non-invasive methodology for detecting aneuploidy. Trial registration number PI21/00283

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Study question Is partial premature ovulation (PPO) detection during the oocyte pick-up (OPU) a sign of poor prognosis in in vitro fertilization (IVF) cycles with own oocytes? Summary answer PPO halves the number of metaphase II oocytes available for an IVF treatment without reducing their quality, demonstrated by unaltered fertilization and top-quality blastocyst rates. What is known already PPO detected during the OPU procedure has not been extensively studied in the literature. This phenomenon may result in a reduction in the number and/or competence of the oocytes retrieved, due to the potential loss of the already expelled oocytes, as well as the likely dominance exerted by the ruptured follicle/s in the rest of the cohort. Despite this, several authors have demonstrated that competent oocytes can be retrieved from these already ruptured follicles, suggesting that oocyte extrusion frequently does not occur after follicle rupture. The potential negative effect exerted in the rest of sibling oocytes remains unknown. Study design, size, duration Retrospective cohort analysis performed in IVIRMA Valencia (Spain), including 8994 cycles of controlled ovarian stimulation (COS) for an IVF treatment with fresh own oocytes, between January 2016 and May 2021. OPU procedures for oocyte cryopreservation, as well as mixed cycles with both fresh and frozen oocytes, were discarded. PPO diagnosis was based on ultrasound visualization of any already formed corpus luteum structure/s, a lower follicular count than expected, and/or free fluid. Participants/materials, setting, methods Female patients undergoing OPU after COS for a fresh IVF treatment. Cycles in which PPO has been detected will be compared with a random, and of the same size, sample without PPO. Mean number of oocytes, metaphase II, fertilized oocytes and top-quality embryos, as well as IVF success rates, will be compared between both groups. Patients’ basal characteristics and COS parameters will be analyzed in order to detect any potential early indicator of PPO. Main results and the role of chance PPO was detected in 123 of the 8994 cycles (1.37%) performed. A random control group of 123 cycles without PPO was selected. Patients’ mean age was 37.6±3.6, with a BMI of 23.3±4.1 kg/m2 and an anti-mullerian hormone of 1.62±1.3 ng/mL. Patient’s basal characteristics and COS parameters were statistically comparable among groups (p > 0.05), except for lower serum estradiol levels (2037.64 vs. 1582.24 pg/mL; p = 0.004) in the PPO group on the last ultrasound prior to OPU. Patients with PPO showed lower aspiration rates (88.95% vs. 55.78% in the PPO gr.), as well as a reduced mean number of oocytes (10.69 vs. 5.68 in the PPO gr.), metaphase II (8.41 vs. 4.33 in the PPO gr.), fertilized oocytes (6.23 vs. 3.26 in the PPO gr.) and top-quality blastocysts (2.77 vs. 1.35 in the PPO gr.) (p = 0.000). In contrast, maturation (80.72% vs. 76.57% in the PPO gr.), fertilization (73.52% vs. 75.18% in the PPO gr.) and top-quality blastocyst rates (44.03% vs. 38.68% in the PPO gr.) were statistically similar between both groups (p > 0.05). Limitations, reasons for caution The main limitations of the present study are its retrospective design and its small sample size, derived from the low frequency of the PPO phenomenon in our clinic. Larger prospective studies should be proposed in order to accurately define the negative impact of PPO in IVF success rates. Wider implications of the findings PPO clearly reduces the number of oocytes available for an IVF treatment, although it does not seem to impair the competence of the remaining cohort. Once PPO is detected, cycle cancellation may not be worth the associated loss of money, time and morale, especially given its low prevalence (around 1%). Trial registration number Not applicable