Abstract Study question Are Prokineticin-1 (PROK1) follicular fluid (FF) levels associated with poor ovarian response (POR) as well as with other well-known biomarkers associated with ovarian reserve status? Summary answer Poor responders present with increased PROK1 FF levels. Also, PROK1 FF levels are negatively correlated with anti-müllerian hormone (AMH) levels and number of oocytes retrieved. What is known already Prokineticin-1 is an angiogenic factor with pleiotropic properties. Considering its role in reproduction, PROK1 mediates significant angiogenic functions in the fetal-maternal interface. Thus, alterations in PROK1 expression and/or function lead to placenta-derived pregnancy complications. Recently published studies also indicate that PROK1 is associated with ovarian function and oocyte competence. However, limited data associating PROK1 with POR are published. This study uniquely investigates possible associations between PROK1 FF levels in POR cases, with stimulation outcome, as well as with FF levels of other factors related to ovarian functionality, namely VEGF, bone morphogenetic protein 15 (BMP-15) and Pigment Epithelium Derived Factor (PEDF). Study design, size, duration This prospective-observational study was collaboratively conducted between November 2020 and July 2021 at Genesis Athens Clinic and at the University of Athens Medical School. A total of 64 patients undergoing IVF treatment were enrolled. The study group comprised of 32 POR patients defined according to the Bologna criteria. The control group consisted of 32 normal responder women undergoing IVF due to tubal factor and/or mild male factor infertility. Patients with other infertility aetiologies were excluded. Participants/materials, setting, methods Participants in both groups received the standard short GnRH-antagonist protocol. Prior to ovarian stimulation, participants were subjected to basic infertility investigation, including antral follicle count and AMH levels evaluation. The FF samples were collected as part of the oocyte retrieval process, then centrifuged and stored at -80 °C till analysis. Follicular fluid levels of PROK1, VEGF, BMP-15 and PEDF were evaluated via ELISA employing commercially available kits. Statistical analysis was performed employing R Programming Language. Main results and the role of chance Significantly higher PROK1 (3229.63 ± 2372.66 vs 1945.95 ± 1408.01 pg/ml; P-value<0.0001), VEGF (2309.63 ± 412.49 vs 2013.22 ± 330.16 pg/ml; P-value=0.006) and lower BMP-15 (434.13 ± 106.38 vs 532.5 ± 108.26 pg/ml; P-value=0.001) levels were recorded in the POR group. No difference was observed regarding PEDF levels (7.43 ± 1.95 vs 8.23 ± 2.54 ng/ml; P-value=0.2). However, the POR group presented with lower PEDF/VEGF ratio (3.33 ± 1.08 vs 4.18 ± 1.41; P-value=0.02), indicating a reduced antioxidant capacity. PROK1 levels were negatively correlated with AMH (P-value=0.04), number of oocytes retrieved (P-value=0.001) and number of MII oocytes (P-value=0.005). BMP-15 was positively correlated with number of oocytes retrieved (P-value<0.001), number of MII oocytes (P-value=0.002), number of normally fertilized zygotes (P-value=0.007), number of cleavage stage embryos (P-value=0.03) and number of blastocysts (P-value=0.04). VEGF was negatively correlated with AMH (P-value=0.004) and number of oocytes retrieved (P-value=0.03). These correlations remained significant when adjusting for ovarian response status. PROK1 levels, with a cut-off value of 2854.25 pg/ml, were able to predict ovarian response status with an area under the curve at 0.64. Sensitivity was 0.55, specificity was 0.88, and accuracy was 0.71. The positive and negative predictive values were 81.82% and 66.67%, respectively. Limitations, reasons for caution Limitations of our study refer to the limited size of the studied population, as well as to the lack of data considering pregnancy outcomes. Moreover, molecular data with regards to the possible role of PROK1 on POR pathogenesis are required. Future studies are needed to verify the results presented herein. Wider implications of the findings Data presented herein indicate that PROK1 FF levels are strongly associated with diminished ovarian reserve and POR. Considering that FF-PROK1 presents with a similar profile with FF-VEGF, we can form the hypothesis that the compromised angiogenesis observed in POR patients leads to PROK1 and VEGF increase via a negative-feedback-loop. Trial registration number Not applicable

Abstract Study question Could embryo accumulation employing mild stimulation cycles prove beneficial for managing patients presenting with poor ovarian response (POR)? Summary answer Embryo accumulation may be an efficient POR management strategy, enabling a higher number and quality cohort of embryos, ultimately improving success results. What is known already It is widely accepted that POR constitutes a challenging condition. The limited oocyte yield associated with POR detrimentally impacts in vitro fertilization (IVF) success rates. Moreover, the documented heterogeneity among POR patients compromises our efforts to successfully address POR, despite the advances noted regarding stimulation protocols employed today. Considering the aforementioned, embryo accumulation following consecutive stimulation cycles has emerged as an alternative management strategy towards increasing the number of available embryos prior to embryo transfer (ET), mimicking normoresponding conditions. However, only few studies have been so far conducted and the need for further data is underlined. Study design, size, duration A single-center retrospective study was conducted in the Centre of Human Reproduction, Genesis-Athens Clinic from January 2015-December 2019. Only patients presenting with POR according to Bologna criteria were included. In total, 610 POR patients were considered eligible and were divided in three groups namely, mild stimulation-fresh ET (150 IUs of gonadotropins) (MILDF), mild stimulation employing embryo accumulation (MILDA), and natural cycle employing embryo accumulation (NATA). Respective comparisons on embryology and pregnancy data are provided. Participants/materials, setting, methods Resulting embryos from the MILDF, MILDA, and NATA groups were cultured up to the cleavage stage and categorized into three groups according to quality, namely top (grade 1), good (grade 2–3) and poor (grade 4–5) (Veeck, 1999). Top and good quality embryos were considered eligible for ET/vitrification. The banking scenario entailed accumulation of at least three embryos, including at least one top quality embryo. Embryo transfers included up to two cleavage stage embryos. Main results and the role of chance Comparing MILDF and MILDA groups, a higher number of available oocytes and embryos was observed in MILDA (2.36±1.15 vs 6.58±1.11; 1.72±1.02 vs 3.51±0.61, P-value<0.001). However, a mean number of 3.90±1.56 oocyte retrievals were required to conclude MILDA compared to MILDF which was concluded following a single oocyte retrieval (P-value<0.001). Cancellation-rate was significantly lower in the MILDA compared to MILDF group (0% vs 18.93%, P-value <0.001). A higher proportion of top quality embryos were transferred in the MILDA group (66.58% vs 43.67%, P-value<0.001). The MILDA group presented with higher positive-HCG (27.89% vs 23.30%, P-value=0.302), clinical-pregnancy (22.11% vs 17.96%, P-value=0.316) and live-birth rates (16.84% vs 14.08%, P-value=0.487). However, these differences were not significant. Comparing MILDA and NATA groups, the MILDA presented with a lower number of required oocyte retrievals and a higher number of oocytes per oocyte retrieval compared with NATA (3.90±1.56 vs 7.15±1.80; 1.95±0.74 vs 0.89±0.20, P-value<0.001). Moreover, the MILDA presented with a higher mean number of resulting embryos (5.20±0.78 vs 4.82±0.88, P-value<0.001). No difference was observed regarding the proportion of the resulting top quality embryos. The MILDA group presented with slightly higher clinical-pregnancy (22.11% vs 20.09%, P-value=0.628) and live-birth (16.84% vs 14.02%, P-value=0.490) rates, however these differences were not significant. Limitations, reasons for caution The retrospective nature of the study constitutes a major limitation. Considering that numerous confounders are inevitable when retrospective data is analyzed, authors employed strict eligibility criteria in an effort to reduce bias. Statistical analysis revealed a well-controlled population, considering that general patients’ characteristics did not differ between the three groups. Wider implications of the findings: Embryo accumulation may constitute an efficient management strategy for POR, as more embryos of better quality are available for ET compared to fresh-IVF-ET. Mild stimulation should be preferred for embryo accumulation instead of natural cycles, as less oocyte retrievals are required. Future studies should be conducted to verify these conclusions. Trial registration number Not applicable

Abstract Study question Can successful implementation of luteal phase oocyte retrieval (LuPOR) following conventional follicular phase oocyte retrieval (FoPOR) be predicted for poor ovarian response (POR) patients? Summary answer Antral follicle count (AFC), number of small follicles recorded in FoPOR, and estradiol (E2) levels on FoPOR and LuPOR trigger days, predict successful LuPOR application. What is known already A second follicular wave in the same menstrual cycle was first observed in domestic animals such as horses and cattle and thenceforth in women. The second follicular wave has been introduced as an encouraging means towards optimizing the context of in vitro fertilization (IVF) success rates for infertile women and especially for POR patients. Double ovarian stimulation coupled with two oocyte retrievals in the same menstrual cycle has been proposed, and encouraging results have been reported. However, the high heterogeneity characterizing POR patients dictates that studies should focus on factors indicating efficient LuPOR application. Study design, size, duration This retrospective observational study included 1688 women diagnosed with POR, undergoing natural IVF cycles between 2012–2020 including two oocyte retrievals in the same menstrual cycle. Patients’ age, body mass index (BMI), number of previous POR incidences, basal hormonal levels, AFC, E2 evaluated on both trigger days and number of small follicles (8–13 mm) were evaluated on their predictive power regarding retrieval of at least one MII oocyte following LuPOR, being regarded as successful LuPOR implementation. Participants/materials, setting, methods A diagnosis of POR according to Bologna criteria served as the inclusion criterion for this single center study. All other infertility etiologies were excluded. Patient dataset was stratified according to age in quantiles. A random 20% of each quantile was employed to validate the model. The remaining 80% was employed to develop this model. The predictive value was determined employing the Area Under the Curve (AUC) of the Receiver Operating Characteristics, employing Youden’s index. Main results and the role of chance Patients’ age, BMI, number of previous failed IVF attempts, basal levels of follicle stimulating hormone (FSH), luteinizing hormone (LH), prolactin and progesterone failed to be predictive of a successful LuPOR as the AUC was below 0.6. AFC with a threshold value of 4.47, was found to be predictive of an effective LuPOR with an AUC of 0.86, sensitivity 0.8, specificity 0.75, and accuracy 0.79. E2 levels evaluated on the FoPOR trigger day, with a threshold value of 232.66 pg/ml, were similarly predictive of an effective LuPOR presenting with an AUC 0.86, specificity 0.75, sensitivity 0.86 and accuracy 0.82. Similarly, E2 evaluated on the LuPOR trigger day, with a threshold value of 200.89 pg/ml, presented with an AUC 0.89, specificity 0.85, sensitivity 0.95 and accuracy 0.92. The number of small follicles during FoPOR also appeared to be predictive of the presence of at least one MII oocyte during LuPOR, with a threshold value of 2.94. The AUC was 0.82, specificity 0.75, sensitivity 0.76 and accuracy 0.75. When combining the above characteristics into a single predictive model the AUC was 0.88, specificity 0.73, sensitivity 0.94 and accuracy 0.89. The positive and negative predictive value of the model were 93.5% and 46.8%, respectively. Limitations, reasons for caution Employment of natural cycles may present as a limitation when examining the value of this study, as the cut-off values reported herein may be altered when stimulation is employed. Since internal validation may be confounded by the fact that this was a single center study, external validation is required. Wider implications of the findings: The clinical end-point of this study reporting back to the practitioner, is the development of a predictive model identifying the optimal POR population for whom LuPOR practice is valuable. The high positive predictive value of this model may assist clinicians in identifying poor responders who will benefit from this approach. Trial registration number Not applicable