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Artificial intelligence applied to time-lapse microscopy may revolutionize embryo selection in IVF by automating data collection and standardizing the assessments. In this context, blastocyst expansion dynamics, although being associated with reproductive fitness, have been poorly studied. This retrospective study (N = 2184 blastocysts from 786 cycles) exploited both technologies to picture the association between embryo and inner-cell-mass (ICM) area in µm2, the ICM/Trophectoderm ratio, and the zona pellucida thickness in µm (zp-T) at sequential blastocyst expansion stages, with (i) euploidy and (ii) live-birth per transfer (N = 548 transfers). A quantitative-standardized-expansion-assay (qSEA) was also set-up; a novel approach involving automatic annotations of all expansion metrics every 30 min across 5 h following blastulation. Multivariate regressions and ROC curve analyses were conducted. Aneuploid blastocysts were slower, expanded less and showed thicker zp. The qSEA outlined faster and more consistent zp thinning processes among euploid blastocysts, being more or as effective as the embryologists in ranking euploid embryo as top-quality of their cohorts in 69% of the cases. The qSEA also outlined faster and more consistent blastocyst expansion and zp thinning dynamics among euploid implanted versus not implanted blastocysts, disagreeing with embryologists’ priority choice in about 50% of the cases. In conclusion, qSEA is a promising objective, quantitative, and user-friendly strategy to predict embryo competence that now deserves prospective validations.

STUDY QUESTION What are the implications of the presence cytoplasmic strings (Cyt-S) and their quantity and dynamics for the pre-implantation development of human blastocysts? SUMMARY ANSWER Cyt-S are common in human embryos and are associated with faster blastocyst development, larger expansion, and better morphological quality. WHAT IS KNOWN ALREADY Cyt-S are dynamic cellular projections connecting inner cell mass and trophectoderm (TE) cells, that can be observed during blastocyst expansion. Their prevalence in human embryos has been estimated to be between 44% and 93%. Data relevant to their clinical implications and role in development are lacking, limited, or controversial. STUDY DESIGN, SIZE, DURATION Retrospective study conducted at a single IVF center between May 2013 and November 2014 and involving 124 pre-implantation genetic testing for aneuploidy cycles in a time-lapse incubator with ≥1 blastocyst biopsied and vitrified (N = 370 embryos assessed). These cycles resulted in 87 vitrified-warmed single-euploid blastocyst transfers. PARTICIPANTS/MATERIALS, SETTING, METHODS ICSI, continuous blastocyst culture (Days 5–7), TE biopsy of fully expanded blastocysts without Day 3 zona pellucida drilling, qPCR to assess uniform full-chromosome aneuploidies, and vitrification were all performed. Only vitrified-warmed euploid single-embryo-transfers were conducted. Blastocyst morphological quality was defined according to Gardner's criteria. The AI-based software CHLOE™ (Fairtility) automatically registered timings from time of starting blastulation (tSB) to biopsy (t-biopsy, i.e. blastocyst full-expansion) as hours-post-insemination (hpi), embryo area (including zona pellucida in µm2), and spontaneous blastocyst collapses. One senior embryologist manually annotated Cyt-S presence, quantity, timings, and type (thick cell-to-cell connections and/or threads). All significant associations were confirmed through regression analyses. All couples', cycles', and embryos' main features were also tested for associations with Cyt-S presence, quantity, and dynamics. MAIN RESULTS AND THE ROLE OF CHANCE About 94.3% of the patients (N = 117/124) had ≥1 embryo with Cyt-S. Out of a total of 370 blastocysts, 55 degenerated between blastulation and full-expansion (N = 55/370, 14.9%). The degeneration rate among embryos with ≥1 Cyt-S was 10.8% (N = 33/304), significantly lower than that of embryos without Cyt-S (33.3%, N = 22/66, P  < 0.01). Of the remaining 315 viable blastocysts analyzed, 86% (N = 271/315; P  < 0.01) had ≥1 Cyt-S, on average 3.5 ± 2.1 per embryo ranging 1–13. The first Cyt-S per viable embryo appeared at 115.3 ± 12.5 hpi (85.7–157.7), corresponding to 10.5 ± 5.8 h (0.5–31) after tSB. Overall, we analyzed 937 Cyt-S showing a mean duration of 3.8 ± 2.7 h (0.3–20.9). Cyt-S were mostly threads (N = 508/937, 54.2%) or thick cell-to-cell connections becoming threads (N = 382/937, 40.8%) than thick bridges (N = 47/937, 5.0%). The presence and quantity of Cyt-S were significantly associated with developmentally faster (on average 6–12 h faster) and more expanded (on average 2700 µm2-larger blastocyst's area at t-biopsy) embryos. Also, the presence and duration of Cyt-S were associated with better morphology. Lastly, while euploidy rates were comparable between blastocysts with and without Cyt-S, all euploid blastocysts transferred from the latter group failed to implant (N = 10). LIMITATIONS, REASONS FOR CAUTION Cyt-S presence and dynamics were assessed manually on seven focal planes from video frames recorded every 15 min. The patients included were mostly of advanced maternal age. Only associations could be reported, but no causations/consequences. Lastly, larger datasets are required to better assess Cyt-S associations with clinical outcomes. WIDER IMPLICATIONS OF THE FINDINGS Cyt-S are common during human blastocyst expansion, suggesting their physiological implication in this process. Their presence, quantity and dynamics mirror embryo viability, and morphological quality, yet their role is still unknown. Future basic science studies are encouraged to finally describe Cyt-S molecular nature and biophysical properties, and Artificial Intelligence tools should aid these studies by incorporating Cyt-S assessment. STUDY FUNDING/COMPETING INTEREST(S) None. TRIAL REGISTRATION NUMBER N/A. [ABSTRACT FROM AUTHOR]

Infertility affects 10%–15% of couples worldwide. Of all infertility cases, 20%–70% are due to male factors. In the past, men with severe male factor (SMF) were considered sterile. Nevertheless, the development of intracytoplasmic sperm injection (ICSI) drastically modified this scenario. The advances in assisted reproductive technology (ART), specifically regarding surgical sperm retrieval procedures, allowed the efficacious treatment of these conditions. Yet, before undergoing ICSI, male factor infertility requires careful evaluation of clinical and lifestyle behavior together with medical treatment. Epidemiologically speaking, women whose male partner is azoospermic tend to be younger and with a better ovarian reserve. These couples, in fact, are proposed ART earlier in their life, and for this reason, their ovarian response after stimulation is generally good. Furthermore, in younger couples, azoospermia can be partially compensated by the efficient ovarian response, resulting in an acceptable fertility rate following in vitro fertilization (IVF) techniques. Conversely, when azoospermia is associated with a reduced ovarian reserve and/or advanced maternal age, the treatment becomes more challenging, with a consequent reduction in IVF outcomes. Nonetheless, azoospermia seems to impair neither the euploidy rate at the blastocyst stage nor the implantation of euploid blastocysts. Based on the current knowledge, the assessment of male infertility factors should involve: (1) evaluation – to diagnose and quantify seminologic alterations; (2) potentiality – to determine the real possibilities to improve sperm parameters and/or retrieve spermatozoa; (3) time – to consider the available “treatment window”, based on maternal age and ovarian reserve. This review represents an update of the definition, prevalence, causes, and treatment of SMF in a modern ART clinic.

Abstract Introduction Several studies suggest that luteinizing hormone (LH) could improve IVF outcome in women of advanced reproductive age by optimizing androgen production. In this review, we assessed the role of recombinant-human LH (r-hLH) and recombinant human follicle stimulating hormone (r-hFSH) co-treatment in ovarian stimulation for assisted reproductive technology in women of advanced reproductive age candidates for assisted reproduction. Material and methods Using a preregistered protocol we systematically searched Medline/PubMed, Scopus and the ISI Web of Science databases to identify randomized controlled trials in which r-hFSH monotherapy protocols were compared with r-hFSH/r-hLH co-treatment in women ≥35 years undergoing fresh IVF cycles. We calculated the pooled odds ratio (OR) for dichotomous data and the weight mean difference (WMD) for continuous data with an associated 95% confidence interval (CI). The meta-analyses were conducted using the random-effect model. P values

Abstract The prolonged lockdown of health services providing high-complexity fertility treatments –as currently recommended by many reproductive medicine entities– is detrimental for society as a whole, and infertility patients in particular. Globally, approximately 0.3% of all infants born every year are conceived using assisted reproductive technology (ART) treatments. By contrast, the total number of COVID-19 deaths reported so far represents approximately 1.0% of the total deaths expected to occur worldwide over the first three months of the current year. It seems, therefore, that the number of infants expected to be conceived and born –but who will not be so due to the lockdown of infertility services– might be as significant as the total number of deaths attributed to the COVID-19 pandemic. We herein propose remedies that include a prognostic-stratification of more vulnerable infertility cases in order to plan a progressive restart of worldwide fertility treatments. At a time when preventing complications and limiting burdens for national health systems represent relevant issues, our viewpoint might help competent authorities and health care providers to identify patients who should be prioritized for the continuation of fertility care in a safe environment.

Recent evidence suggests that follicular development occurs in a wave-like model during the ovarian cycle, where up to three cohorts of follicles are recruited to complete folliculogenesis. This understanding overtakes the previous dogma stating that follicles grow only during the follicular phase of the menstrual cycle. Therefore, in in vitro fertilization (IVF), novel protocols regarding ovarian stimulation have been theorized based on the use of gonadotrophins to prompt the growth of antral follicles at any stage of the menstrual cycle. These unconventional protocols for ovarian stimulation aim at a more efficient management of poor-prognosis patients, otherwise exposed to conflicting outcomes after conventional approaches. DuoStim appears among these unconventional stimulation protocols as one of the most promising. It combines two consecutive stimulations in the follicular and luteal phases of the same ovarian cycle, aimed at increasing the number of oocytes retrieved and embryos produced in the short time-frame. This protocol has been suggested for the treatment of all conditions requiring a maximal and urgent exploitation of the ovarian reserve, such as oncological patients and poor responders at an advanced maternal age. At present, data from independent studies have outlined the consistency and reproducibility of this approach, which might also reduce the drop-out between consecutive failed IVF cycles in poor-prognosis patients. However, the protocol must be standardized, and more robust studies and cost-benefit analyses are needed to highlight the true clinical pros and cons deriving from DuoStim implementation in IVF.

STUDY QUESTION What are the clinical pregnancy and live birth rates in women who underwent up to two more euploid blastocyst transfers after three failures in the absence of another known factor that affects implantation? SUMMARY ANSWER The fourth and fifth euploid blastocyst transfers resulted in similar live birth rates of 40% and 53.3%, respectively, culminating in a cumulative live birth rate of 98.1% (95% CI = 96.5–99.6%) after five euploid blastocyst transfers. WHAT IS KNOWN ALREADY The first three euploid blastocysts have similar implantation and live birth rates and provide a cumulative live birth rate of 92.6%. STUDY DESIGN, SIZE, DURATION An international multi-center retrospective study was conducted at 25 individual clinics. The study period spanned between January 2012 and December 2022. A total of 123 987 patients with a total of 64 572 euploid blastocyst transfers were screened for inclusion. PARTICIPANTS/MATERIALS, SETTING, METHODS Patients with a history of any embryo transfer at another clinic, history of any unscreened embryo transfer at participating clinics, parental karyotype abnormalities, the use of donor oocytes or a gestational carrier, untreated intracavitary uterine pathology (e.g. polyp, leiomyoma), congenital uterine anomalies, adenomyosis, communicating hydrosalpinx, endometrial thickness <6 mm prior to initiating of progesterone, use of testicular sperm due to non-obstructive azoospermia in the male partner, transfer of an embryo with a reported intermediate chromosome copy number (i.e. mosaic), preimplantation genetic testing cycles for monogenic disorders, or structural chromosome rearrangements were excluded. Ovarian stimulation protocols and embryology laboratory procedures including trophectoderm biopsy followed the usual practice of each center. The ploidy status of blastocysts was determined with comprehensive chromosome screening. Endometrial preparation protocols followed the usual practice of participating centers and included programmed cycles, natural or modified natural cycles. MAIN RESULTS AND THE ROLE OF CHANCE A total of 105 (0.085% of the total population) patients met the criteria and underwent at least one additional euploid blastocyst transfer after failing to achieve a positive pregnancy test with three consecutive euploid blastocyst transfers. Outcomes of the fourth and fifth euploid blastocyst transfers were similar across participating centers. Overall, the live birth rate was similar with the fourth and fifth euploid blastocysts (40% vs 53.3%, relative risk = 1.33, 95% CI = 0.93–1.9, P value = 0.14). Sensitivity analyses excluding blastocysts biopsied on Day 7 postfertilization, women with a BMI >30 kg/m2, cycles using non-ejaculate or donor sperm, double-embryo transfer cycles, and cycles in which the day of embryo transfer was modified due to endometrial receptivity assay test result yielded similar results. Where data were available, the fourth euploid blastocyst had similar live birth rate with the first one (relative risk = 0.84, 95% CI = 0.58–1.21, P  = 0.29). The cumulative live birth rate after five euploid blastocyst transfers was 98.1% (95% CI = 96.5–99.6%). LIMITATIONS, REASONS FOR CAUTION Retrospective design has its own inherent limitations. Patients continuing with a further euploid embryo transfer and patients dropping out from treatment after three failed euploid transfers can be systematically different, perhaps with regard to ovarian reserve or economic status. WIDER IMPLICATION OF THE FINDINGS Implantation failure seems to be mainly due to embryonic factors. Given the stable and high live birth rates up to five euploid blastocysts, unexplained recurrent implantation failure should have a prevalence of <2%. Proceeding with another embryo transfer can be the best next step once a known etiology for implantation failure is ruled out. STUDY FUNDING/COMPETING INTEREST(s) None. TRIAL REGISTRATION NUMBER N/A. [ABSTRACT FROM AUTHOR]

The POSEIDON (Patient-Oriented Strategies Encompassing IndividualizeD Oocyte Number) criteria were developed to help clinicians identify and classify low-prognosis patients undergoing assisted reproductive technology (ART) and provide guidance for possible therapeutic strategies to overcome infertility. Since its introduction, the number of published studies using the POSEIDON criteria has increased steadily. However, a critical analysis of existing evidence indicates inconsistent and incomplete reporting of critical outcomes. Therefore, we developed guidelines to help researchers improve the quality of reporting in studies applying the POSEIDON criteria. We also discuss the advantages of using the POSEIDON criteria in ART clinical studies and elaborate on possible study designs and critical endpoints. Our ultimate goal is to advance the knowledge concerning the clinical use of the POSEIDON criteria to patients, clinicians, and the infertility community.

Despite the considerate progress to which assisted reproduction technology (ART) has been subject since 1978, some issues remain unresolved. Notably, the clinical management of patients with a poor ovarian response is still a challenge in everyday practice, frustrating to both the patient and the fertility expert. Poor ovarian responders (PORs) embody 9–24% of patients undergoing ovarian stimulation, meaning that up to one in four patients conceals a poor reproductive prognosis. The last decade has witnessed the attempts of the medical community to standardize diagnosis of POR with the developing of the Bologna Criteria and the subsequent evolution of the low prognosis patient elaborated in the POSEIDON classification. The aim of this article is to summarize all evidence concerning etiology and management of poor ovarian response, including the most recent advances and future prospects in this regard.

Research Question: Does reproductive outcome differ among the various subgroups of poor ovarian responders according to the Bologna criteria?Design: This was a retrospective, cohort study including poor ovarian responders according to Bologna criteria, undergoing an ICSI cycle from January 2011 until December 2017. Patients were divided into four groups: (1) age ≥ 40 years and abnormal ovarian response test, (2) age ≥ 40 years, abnormal ovarian reserve test and one previous poor response to stimulation, (3) age ≥ 40 years and one previous poor response, (4) abnormal ovarian reserve test and one previous poor response.Result(s): Overall, 846 cycles in 706 Bologna poor ovarian responders were included: 310 cycles in group 1, 169 in group 2, 52 in group 3, and 315 in group 4. There were significant differences in age, antral follicle count, antimüllerian hormone, cycle cancellation rates, and number of retrieved oocytes between the four groups. Live birth and cumulative live birth rate differed significantly between groups and were highest in Group 4 [Live birth rate: 7.4% (1) vs. 4.1% (2) vs. 5.8% (3) vs. 13.4% (4), p = 0.001 and Cumulative live birth rate: 8.3% (1) vs. 4.1 % (2) vs. 9.6% (3) vs. 16.8% (4) p < 0.001]. The multivariate GEE analysis revealed that the number of MIIs and the Bologna criteria pattern were the variables which were significantly associated with cumulative live birth rate.Conclusion(s): Poor ovarian responders represent a heterogeneous population. The young subpopulation has a better clinical prognosis in terms of fresh and cumulative live birth rate.

This multicenter study evaluated the reliability of the recently published ART calculator for predicting the minimum number of metaphase II (MII) oocytes (MIImin) to obtain at least one euploid blastocyst in patients undergoing in vitro fertilization/intracytoplasmic sperm injection (IVF/ICSI). We used clinical and embryonic retrospective data of 1,464 consecutive infertile couples who underwent IVF/ICSI with the intention to have preimplantation genetic testing for aneuploidy. The validation procedure followed a stepwise approach. Firstly, we assessed the distribution of euploid blastocysts per patient and found that it followed a negative binomial distribution. Secondly, we used generalized linear models and applied the Lasso procedure–including MII oocytes to adjust the data–to select the factors predicting the response variable “euploid blastocyst.” Third, a logistic regression model–fit to the binomial response euploid (yes/no) for each MII oocyte–was built using the relevant factors. The observational unit was the “woman” whereas the response was the pair (m, n), where n is the number of retrieved MII oocytes and m the corresponding number of euploid blastocysts. The model was internally validated by randomly splitting the data into training and validation sets. The R-squares (~0.25) and the area under the ROC curve (~0.70) did not differ between the training and validation datasets. Fourth, mathematical equations and the calculated probabilities generated by the validation model were used to determine the MIImin required for obtaining at least one euploid blastocyst according to different success probabilities. Lastly, we compared the fittings generated by the validation model and the ART calculator and assessed the predictive value of the latter using the validation dataset. The fittings were sufficiently close for both the estimated probabilities of blastocyst euploid per MII oocyte (r = 0.91) and MIImin (r = 0.88). The ART calculator positive predictive values, i.e., the frequency of patients with at least one euploid blastocyst among those who achieved the estimated MIImin, were 84.8%, 87.5%, and 90.0% for 70%, 80%, and 90% predicted probabilities of success, respectively. The ART calculator effectively predicts the MIImin needed to achieve at least one euploid blastocyst in individual patients undergoing IVF/ICSI. The prediction tool might be used for counseling and planning IVF/ICSI treatments.