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Methods: This is a cohort study, conducted at a university-based reproductive medicine center and private reproductive medicine center that aimed to evaluate granulosa cumulus cell gene expression in the insulin signaling pathway in Polycystic Ovary Syndrome (PCOS) patients undergoing in vitro fertilization (IVF) treatment and to compare the cumulus gene expression between normal weight and obese women without clinical insulin resistance. Fifteen PCOS patients, nine normal weight patients and six obese patients presenting normal HOMA IR (Homeostasis Model Assessment–Insulin Resistance), participated. Patients underwent oocyte retrieval for IVF and after the procedure, granulosa cumulus cells were removed from the oocytes for RNA extraction. Quantitative polymerase chain reaction (PCR) array analysis of 84 genes from insulin signaling pathway was conducted. The results were expressed as fold up- or fold down-expression in obese patients compared with normal weight patients. Any fold change ⩾3 or ⩽3 and any p ⩽ 0.05 were considered statistically significant. Results: There were 10 genes that were overexpressed in obese compared with normal weight women, BCL2L1, BRAF, CBL, DOK1, FBP1, FRS2, MTOR, PCK2, RPS6KA1, and SORBS1, that had a fold change ⩾3 and p ⩽ 0.05. Discussion: In the obese group, the overexpressed genes are mainly responsible for the proliferation and differentiation of cumulus cells during oocyte maturation, insulin resistance, apoptosis regulation, and glucose metabolism during early embryogenesis, suggesting that in the follicular environment, insulin resistance is present even in the absence of clinical signs. Conclusion: Together, our findings and the related literature suggest that those alterations may be associated with the worse prognosis of follicular development and oocyte maturation observed in PCOS obese women.

Preeclampsia (PE) is a hypertensive disease of pregnancy-associated with placental cell death and endoplasmic reticulum (ER) stress. It is unknown whether systemic factors aggravate placental dysfunction. We investigated whether serum factors in pregnant women with PE activate ER stress and unfolded protein responses (UPRs) in placental explants and trophoblast cells lineage. We cultured placental explants from third-trimester term placentas from control non-preeclamptic (NPE) pregnant women with serum from women with PE or controls (NPE). In PE-treated explants, there was a significant increase in gene expression of GADD34, CHOP, and SDF2. At the protein level, GRP78, SDF2, p-eIF2α, and p-eIF2α/eIF2α ratio were also augmented in treated explants. Assays were also performed in HTR8/SV-neo trophoblast cell line to characterize the putative participation of trophoblast cells. In PE serum-treated protein levels of p-eIF2a and the ratio p-elF2 α/elF2α increased after 12 h of treatment, while the gene expression of GADD34, ATF4, and CHOP was greater than control. Increased expression of SDF2 was also detected after 24 h-cultured HTR8/SV-neo cells. PE serum increased sFLT1 gene expression and decreased PlGF gene expression in placental explants. Morphologically, PE serum increased the number of syncytial knots and reduced placental cell metabolism and viability. Analysis of the serum of pregnant women with PE through Raman spectroscopy showed changes in amino acids, carotenoids, lipids, and DNA/RNA, which may be associated with the induction of ER stress found in chorionic villi treated with this serum. In conclusion, this study provides evidence that the serum of pregnant women with PE may impact placental villi changing its morphology, viability, and secreted functional factors while triggers ER stress and an UPR. The differences between PE and control sera include molecules acting as inducing factors in these processes. In summary, the results obtained in our assays suggest that after the development of PE, the serum profile of pregnant women may be an additional factor that feeds a continuous imbalance of placental homeostasis. In addition, this study may expand the possibilities for understanding the pathogenesis of this disorder.

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Study question Does the diagnosis of mosaicism affect ploidy rates across different providers offering preimplantation genetic testing for aneuploidies (PGT-A)? Summary answer Our analysis of 36395 blastocyst biopsies across 8 genetic testing laboratories revealed that euploidy rates were significantly higher in providers reporting low rates of mosaicism. What is known already Diagnoses consistent with chromosomal mosaicism have emerged as a third category of possible options for embryo ploidy outcomes in PGT-A. However, diagnosing mosaicism using current PGT-A platforms remains hindered by several biological and technical factors. This has led to substantial variability in mosaicism rates amongst genetic testing laboratories. Furthermore, reservations regarding the clinical value of diagnosing mosaicism have led to varying practices in reporting mosaic calls amongst providers. Critically, it remains unknown whether these differences impact the number of euploid embryos available for transfer. Ultimately, this may significantly affect clinical outcomes, with important implications for PGT-A patients. Study design, size, duration Retrospective, international, multicenter cohort study of 10875 PGT-A cycles conducted between October 2015 and October 2021. A total of 18 IVF centers associated with 8 PGT-A providers, across 5 countries and 3 continents participated in the study, which included 36395 blastocysts, tested using trophectoderm biopsy and next generation sequencing (NGS). Both autologous and donation cycles were assessed. Preimplantation genetic testing for structural rearrangements (PGT-SR) cycles were excluded from the analysis. Participants/materials, setting, methods Ploidy rates were analyzed using multilevel mixed linear regression. Providers were categorized (A to H), with the most frequent provider used as the reference for statistical analysis. Analyses were adjusted for maternal age, paternal age, donor status, number of embryo biopsied and day of biopsy, as appropriate. The overall significance of categorical variables in the regression models was tested using a Chi-squared test. P-values <0.05 were considered significant. Data analysis was performed using STATA, v.15.0. Main results and the role of chance The mean maternal age(+SD) across all providers was 36.9(±5.1). As expected, maternal age and day of biopsy had a significant impact on euploidy rates (p < 0.0001). Mosaicism rates were associated with PGT-A provider and independent of all other parameters (maternal age, paternal age, donor status, number of embryos biopsied and day of biopsy). Out of the 8 providers, 7 reported chromosomal mosaicism. Amongst these 7 providers, the rate of mosaic calls varied from 2.9% to 23.9%. After adjusting for confounders, two providers reported significantly higher mosaicism rates compared to the reference (4.2%): Provider-C 10.4% (OR = 2.43, 95%CI: 1.84-4.25) and Provider-F 23.9% (OR = 4.47, 95%CI: 2.92-6.86), while euploidy and aneuploidy rates did not differ. Conversely, the chance of diagnosing mosaicism was lower in Provider-B (OR = 0.34, 95%CI: 0.22-0.54) and Provider-E (OR = 0.59, 95%CI: 0.38-0.90). Here, aneuploidy rates were comparable to the reference, yet the chance of diagnosing a euploid embryo was significantly higher: Provider-B (OR = 2.38, 95%CI: 1.87-3.03) and Provider-E (OR = 1.62, 95%CI: 1.28-2.05). Compared to the reference, euploidy rates were also higher when mosaicism was not reported: 53.5% vs. 44.2% (OR = 2.04, 95% CI: 1.60-2.59). Moreover, the chance of having at least one euploid blastocyst available for transfer significantly increased when mosaicism was not diagnosed (OR = 1.30, 95%CI: 1.13-1.50). Limitations, reasons for caution Due to the retrospective nature of the study, associations can be ascertained, however causality cannot be established. Certain parameters were not available in the dataset, therefore full elucidation of all potential confounders accounting for the variability may not be possible. Wider implications of the findings Our findings highlight the significant impact of the genetic testing provider on PGT-A results. We demonstrate that reporting mosaicism primarily comes at the expense of euploid diagnoses, raising concerns regarding the accuracy of mosaicism predictions and their impact on clinical outcomes. Moving forward, greater standardization amongst providers will be essential. Trial registration number NA

RESULTS: Websites were classified as: 43% hospital based, 12% fertility clinic based, 5% other clinic based, 33% society/association based, and 7% government based. The mean total DISCERN score was 44 AE 12 (maximum score 80). 75% (45/60) of websites had clear aims or achieved their aims (scores of 4 or 5 [good]), but only 15% (9/60) described areas of clinical uncertainty. 25% (15/60) described the benefits of treatments, but only 5% (3/ 60) described the risks of treatments. 72% (43/60) provided unbiased information. 68% (41/60) websites made it clear that there was more than one possible treatment choice. 27% (16/60) of websites encouraged shared decision making. Overall, 60% (36/60) of websites were ''poor quality'' (score 1-2-3) on the final question of the DISCERN instrument. Only 4/60 (6.7%) websites met all four JAMA benchmark criteria. The mean Dale-Chall score was 9.53 +/-1.30, indicating a college or graduate degree level of readability. The mean Flesh Reading Ease index was 34.01 +/-16.26, indicating a graduate degree level of readability. 20% (12/60) of websites were HONcode certified.CONCLUSIONS: Websites on ''male infertility'' are of low quality, and only 6.7% met JAMA benchmark criteria. Minimal information on treatments was present, with only 25% of websites describing treatment benefits, but only 5% describing treatment risks. Only 15% of websites described areas of clinical uncertainty. Despite that these websites were written at a college to graduate degree level of reading, only 27% encouraged shared decision making. Reassuringly, most of these websites were hospital based, and 72% provided unbiased information. Patients should be cautioned that incomplete and potentially biased information on male infertility is prevalent online.

Study question How informative is the score grade of KIDScore version 3 for day 5 blastocyst for clinical pregnancy in biopsied and non-biopsied embryos? Summary answer Potential clinical pregnancy is predicable according to score grades (above 7.0), regardless the use of PGT-A, in blastocysts on day 5. What is known already Time-lapse technology has promoted, along with the use of artificial intelligence (A.I.), a new spectrum of tools to improve embryo selection. Several software and algorithms have been launched in ART field in the last years, with the perspective of providing a substantial boost in IVF outcomes. KIDScore is one of these new tools, developed based on morphology and morphokinetics of embryo development with known clinical outcome and validated with transfer of blastocyst on day 3 or 5. Yet, it is highly recommended an in-house validation of any A.I. tool before it started to be apply in clinical decisions. Study design, size, duration Retrospective cohort study in a single private IVF center. Positive or negative clinical pregnancy (fetal heartbeat and gestational sac presence/absence) record of patient’s autologous and donated cycles using fresh and frozen oocytes, with or without PGT-A embryos transfers using the Embryocope® Plus incubator, that underwent single embryo transfers (total sET, n = 415; euploid = 228, non-biopsied = 187) of blastocysts developed on day 5 were included. Biochemical pregnancy and miscarriage were excluded of this analysis. Participants/materials, setting, methods Negative and positive clinical pregnancy KIDScoreTMDay 5’s were stratified in three subgroups, according to V3 score intervals: subgroup 1: range between 1.0-3.9 (n = 29), subgroup 2: 4.0-6.9 (n = 154) and subgroup 3: 7.0-9.9 (n = 232). sET of euploid embryos (n = 228) were also analyzed in the described subgroups (subgroup 1: n = 17; subgroup 2: n = 93 and subgroup 3: n = 118, respectively). For the analysis, Mann-Whitney, Chi-square and Fisher tests were used for statistical analysis, values of p < 0.05 were considered significant. Main results and the role of chance Maternal age between overall positive and negative pregnancies were similar (38,48±3,86 versus 38,75±3,83,p = 0,3573). When comparing score subgroups, overall positive clinical pregnancy rates were significant different [subgroup 1: 20.7% (6/29); subgroup 2: 43.5% (67/154); subgroup 3: 63.8% (148/232),p < 0.0001]. When analyzing subgroup 1 versus subgroup 2 there was also a difference in positive clinical pregnancy (p = 0.023) and subgroup 3 also showed a higher rate in clinical pregnancy when compared to subgroup 1 and 2 together (scores from 1.0 to 6.9,p < 0.0001). Analyzing only euploid embryos, the results on positive clinical pregnancy were also significant different between subgroups [subgroup 1: 35.3% (6/17); subgroup 2: 45.2% (42/93); subgroup 3: 61.0% (72/118),p = 0,024, and subgroup 1 + 2 versus subgroup 3,p = 0,0115]. Maternal age between positive and negative clinical pregnancies in PGT-A cycles were similar (37,81±1,61 versus 38,38±3,25,p = 0,069). Analyzing only non-biopsied embryos, the results on positive clinical pregnancy were also significant different between subgroups [subgroup 1: 0.0% (0/12); subgroup 2: 41.0% (25/61); subgroup 3: 66.7% (76/114),p = 0,0343, and subgroup 1 + 2 versus subgroup 3,p < 0.0001]. Maternal age between positive and negative clinical pregnancies in non-biopsied cycles were also similar (39,40±4,75 versus 39,22±4,43,p = 0,7816). Positive clinical pregnancy in subgroup 3 were similar in biopsied and non-biopsied subgroups (61% versus 66.7%,p = 0.4133). Limitations, reasons for caution The retrospective nature and low data of subgroup 1 (1.0-3.9 score), since they naturally are the last option to be chosen for transfer. Wider implications of the findings Differences on positive clinical pregnancy between subgroups (mainly scores greater than 7.0) reinforce the use of A.I. as a complementary tool for embryo selection. Interestingly, positive clinical pregnancy in 7.0-9.9 subgroup were similar in euploid and non-biopsied embryos, strengthening another potential application of A.I. in transposing embryo aneuploidy barrier. Trial registration number Not Applicable

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