Study question Is there an association between additional subcutaneous progesterone (a-sP) during luteal phase support for vitrified-warmed euploid blastocyst transfers and the clinical outcomes? Summary answer a-sP was mainly administered to worse-prognosis patients:positive-pregnancy-test (PPT), but also biochemical-pregnancy-loss (BPL) rates were lower than control, thereby involvingsimilar live-birth-rate (LBR) per transfer. What is known already Frozen-embryo-transfer (FET) implementation is increasing worldwide, and luteal-phase-support is a hot-topic. Serum/uterine progesterone levels are key in eliciting immunological tolerance and uterine quiescence, while supporting the processes underlying embryo implantation. Yet, a consensus is missing on progesterone administration (formulation, route, dosage, and duration). Endometrial biopsy studies showed increased progesteroneconcentration after vaginal administration, while sP has excellent pharmacokinetics with faster peak serum concentration.Combining both administration routes might compensate cases ofpoor vaginal absorption and contribute to reaching adequate systemic/uterine levels. This synergy might be beneficial especially in RIF-patients, subject to lower LBRs also in the context of euploid ET. Study design, size, duration Retrospective analysis of 775 vitrified-warmed euploid blastocysttransfers conducted January2020-March2021. Three of 12 gynecologists administrated a-sP as luteal phase support mostly to patients with a worse reproductive history (i.e., longer duration of infertility and/or higher number of previous failed IVF). We assessed the clinical outcomes (PPT, BPL, miscarriage and LBR) in the group of patients who used a-SP (N = 128) versus the control(N = 647), adjusting for confounders in logistic regression analyses. Participants/materials, setting, methods Only euploid non-mosaic blastocysts were transferred. All patients had normal uterine cavities and thyroid function. Endometrial preparation was performed with either hormone-replacement-therapy (estradiol valerate 6mg/die plus vaginal micronized progesterone 800mg/die) or modified-natural-cycle (hCG administration plus vaginal micronized progesterone 400mg/die). In the a-sP group, the supplementation was started 3 days before FET. In case of pregnancy, the therapy was continued until the 8thweek. Main results and the role of chance The patients in the control and a-sP groups were similar for oocyte age (37.6±2.7yr versus 37.5±2.7yr), age at ET (37.7±3.2yr versus 37.7±3.4yr), and body-mass-index (22.2±2.4 versus 22.0±2.8yr). Conversely, the patients in the a-SP group experienced longer duration of infertility (3.5±1.9yr in the control versus 4.1±2.6yr, p = 0.05), and had already undergone ≥1 previous IVF cycle (37%) and ≥2 failed ETs (22%) more frequently than the control (27% and 12%, respectively;p=0.05). The endometrial preparation protocol was similar in the two groups (76% hormone-replacement-therapy and 24% modified-natural-cycle). Also, blastocyst quality and day of transfer were similar. In the study period, the prevalence of a-sP administration increased from first to second-third ETs (13% to 29%). The confounders identified on the clinical outcomes were blastocyst quality, day of transfer and consecutive number of ET. Therefore, these features were included in multivariate logistic regressions. PPT rates were 61.1% (N = 395/647) and 49.2% (N = 63/128, p = 0.01) in the control and a-SP groups, respectively (multivariate-OR:0.65, 95%CI 0.44-0.97;adjusted-p=0.04). BPL rates were 9.1% (N = 36/395) and 1.6% (N = 1/63, p = 0.04), respectively (multivariate-OR:0.14, 95%CI 0.02-0.99;adjusted-p=0.05). Miscarriage rates were 14.2% (N = 51/359) and 6.5% (N = 4/62;p=0.1), respectively (multivariate-OR:0.4, 95%CI 0.14-1.16;adjusted-p=0.1). Lastly, LBRs were 47.6% (N = 308/647) and 45.3% (N = 58/128), respectively (multivariate-OR:1.04, 95% 0.7-1.55;adjusted-p=0.8). Limitations, reasons for caution Retrospective analysis. Progesterone levels are not routinely assessed in our clinical practice. A larger prospective study only in poor prognosis patients is required to assess the putative benefit of a-sP. Wider implications of the findings About 30% of patients undergoing FET suffer from inadequate progesterone levels, possibly impacting the clinical outcomes, yetscarcely detectable through its serum levels. In these women, vaginal progesterone could be insufficient, and a-sP can act as a rescue strategy against reduced endometrial receptivity via lower BPL. Trial registration number Not applicable

Study question Is double stimulation in the same ovarian cycle (DuoStim) a valuable strategy to improve the chance of success in couples carrier of monogenic conditions? Summary answer DuoStim is advantageous in patients indicated for preimplantation genetic testing for monogenic diseases and aneuploidy testing (PGT-M/-A) and obtaining ≤5 blastocysts after the I-stimulation. What is known already PGT-M is a well-established strategy to identify unaffected blastocysts in a cohort of embryos produced from couples carrier of single gene disorders. When PGT-M is conducted in poor prognosis and/or advanced maternal age (POR/AMA) patients, also aneuploidy-testing among unaffected blastocysts is suggested on a single biopsy. The Poseidon group stated that retrieving an adequate number of oocytes to produce ≥1 euploid blastocyst should be considered the main goal of controlled-ovarian-stimulation (COS). This task is even more complex when PGT-M is required in POR/AMA patients. Therefore, lately we started suggesting DuoStim to these couples to maximize their chance of success. Study design, size, duration Multicenter case series (2016-2021). Comprehensive description of IVF outcomes derived from DuoStim application in POR/AMA patients undergoing PGT-M. Aneuploidy-testing was also conducted on unaffected blastocysts. All results were compared between the two stimulations in the same ovarian cycle, and we reported the contribution of II-stimulations to higher chances of success. Participants/materials, setting, methods GnRH-antagonist COS was performed with recombinant-gonadotrophins and agonist-trigger. Based on the expected risk each embryo would be affected-aneuploid, DuoStim was suggested to all patients obtaining ≤5 blastocysts (day5-7 from I-retrieval). 61 patients accepted and underwent II-stimulations with the same protocol. Only ICSI, trophectoderm-biopsy, qPCR, and vitrified-warmed unaffected-euploid single-blastocyst-transfer(s) were conducted. Main results and the role of chance 90 patients (36.7±3.7yr) indicated to PGT-M/-A and obtaining ≤5 blastocysts after I-stimulations were suggested starting a II-stimulation in the same ovarian cycle. Among them, 61 accepted (67%). 7.2±4.4 and 7.4±4.2 cumulus-oocyte-complexes were retrieved after I- and II-stimulations, respectively (p = 0.7). 5.1±3.0 and 5.8±3.4 were metaphase-II oocytes (p = 0.2), resulting in 75±21% and 77±25% maturation-rates (p = 0.6). 3.6±2.1 and 4.6±2.9 2PN-zygotes were obtained (p = 0.04), resulting in 75±27% and 81±20% fertilization-rates (p = 0.19). 1.6±1.3 and 2.2±1.8 blastocysts were obtained (p = 0.04), resulting in 43±32% and 46±32% blastulation-rates (p = 0.6). Lastly, 0.5±0.7 and 0.7±1.0 blastocysts were diagnosed unaffected-euploid (p = 0.3), resulting in 35±40% and 30±36% transferable blastocyst rates per biopsied embryo, respectively (p = 0.53), and identical 10±16% transferable blastocyst rates per metaphase-II oocyte in both groups (p = 0.9). 23 (38%) and 24 (39%) patients obtained ≥1 transferable blastocyst after the I- and II-simulation, respectively. Overall, 36 (59%) patients obtained ≥1 transferable blastocyst thanks to DuoStim. To date, 57 (93%) cycles were concluded, and the cumulative live birth rate (CLBR) was 37% (N = 21/57). The patients with a LB have 1.7 surplus transferable blastocysts, and 3 patients already delivered 2 singleton healthy-LBs. The CLBR among patients undergoing the conventional strategy was 21%, no surplus transferable blastocyst is available, and no patient delivered >1 LB. Limitations, reasons for caution Observational case series based on real-life data. Of note, the adoption of DuoStim (or any other oocyte/embryo accumulation strategy) in PGT-M/-A is valuable also to amortize the costs of PGT-M set-up and genetic testing through a larger number of blastocysts. In this context, cost-effectiveness analyses in different settings are desirable. Wider implications of the findings DuoStim is a fully-personalized strategy advisable any time lower chance of success and higher treatment costs could be balanced by a larger number of blastocysts produced in a short time-frame. Adopting GnRH-antagonist protocols in these patients allows to suggest DuoStim even in progress, based on the embryological outcomes after I-stimulations. Trial registration number Not applicable

Study question Can progestins be used to prevent spontaneous LH surge during ovarian stimulation (OS) without affecting the euploid blastocyst rate (EBR) per metaphase-II (MII) oocytes? Summary answer Progestin Primed Ovarian Stimulation (PPOS) (conducted with norethisterone acetate) and conventional-OS with GnRH-antagonist show similar EBR per MII-oocytes What is known already PPOS is a novel OS protocol based exogenous progesterone to inhibit LH surge and spontaneous ovulation as an alternative to GnRH analogues. The rationale of its use is that multiple follicular waves arise during the ovarian cycle and no spontaneous ovulation occurs during the luteal phase. PPOS effectiveness and safety have been supported lately in the literature. A recent study reported similar number of euploid blastocysts after PPOS (conducted with medroxyprogesterone acetate) versus conventional-OS with GnRH-antagonist. Study design, size, duration Case-control study involving advanced-maternal-age women undergoing ICSI with PGT-A at two private IVF centers (May-2017 to February-2020). 80 PPOS women were matched with 160 control based on maternal-age and ovarian reserve markers. Patients with poor-ovarian-reserve (AFC<3), premature-ovarian-failure, III-IV stage endometriosis, ovarian cyst, severe-male-factor, PGT-SR or PGT-M were excluded. EBR per MII-oocytes was the primary outcome. All other embryological and clinical outcomes were reported. Participants/materials, setting, methods Both groups underwent recombinant-FSH OS with GnRH-agonist ovulation trigger; the only difference was that norethisterone acetate 10mg/day was administered orally to PPOS patients starting from the second day of the menstrual cycle until trigger. The control group, instead, underwent conventional antagonist protocol. The laboratory procedures were similar in both groups: ICSI, blastocyst culture, trophectoderm biopsy, comprehensive-chromosome-testing to report non-mosaic aneuploidies and vitrified-warmed euploid single-embryo-transfers (SETs). Main results and the role of chance The mean fertilization rate per MII-oocytes and blastulation rate per 2PN-zygotes were similar among PPOS and control (75.5%±21.8% versus 71.7±20.7%, p = 0.1; and 56.5%±27.7% versus 53.2%±27.5%, p = 0.4, respectively). No difference was reported for the EBR per MII-oocytes among PPOS and control (15.4%±19.8% versus 14.8%±18.2%, p = 0.9). No difference was reported among the biopsied blastocysts in terms of morphological quality and day of development. A total of 47 and 86 SETs were conducted in the two groups. There was no difference in the live-birth-rates (LBR) per SET in the two groups (N = 19/47, 40.4%, 95%CI 26.7-55.7 versus N = 35/86, 40.7%, 95%CI 30.4-51.8, p = 0.9), nor in the miscarriage rate per clinical pregnancy (N = 1/20, 5%, 95%CI 0.3-26.9 versus N = 3/36, 8.3%, 95%CI 2.2-25.6, p = 0.9). At last, the cumulative-LBR among concluded cycles (i.e., LB achieved or no transferable/all transferred euploid blastocysts) was also similar (N = 19/68, 27.9%, 95%CI 18.1-40.3 versus N = 31/132, 22.0%, 95%CI 15.4-30.2, p = 0.4). Limitations, reasons for caution The main limitation is the retrospective and non-randomized design. More data are required, especially for follicle recruitment, oocyte yield, gestational and perinatal outcomes. Wider implications of the findings Norethisterone acetate-based PPOS protocol has no impact on oocyte competence when compared to conventional GnRH-antagonist OS. These promising data support further investigation on PPOS, especially in view of its reduced cost, and putative increase in patient compliance and decrease in discomfort. Trial registration number none