Changes in Sperm Parameters with Time in Men with Normal and Abnormal Baseline Semen Analysis.

The association between paternal age and sperm quality in the population level has been previously studied. Only limited data exists regarding the intra-personal variations in semen parameters among fertile and infertile men over time. We aimed to assess trends over time in semen parameters among men with normal and abnormal baseline sperm parameters and investigate potential risk factors for sperm quality deterioration. This retrospective cohort study was conducted at a university-affiliated medical center in vitro fertilization (IVF) unit. Patients with at least two semen analyses (SA) performed > 1 year apart, with the last SA done between 2017 and 2021, were included. The study consisted of two main analyses-comparison of intra-patient's sperm parameters changes in men with normal and abnormal baseline SA (BSA) and analysis of risk factors for developing abnormal semen parameters over time in men who had normal BSA parameters. This study included a total of 902 men assessed for infertility with normal and abnormal BSA. The average time interval between tests was 1015 days (range 366-7709 days). Among individuals with normal BSA, there was a mild decline in most parameters-concentration (- 6.53 M/ml), motility (- 7.74%), and total motile count (TMC) (- 21.80 M) (p < 0.05 for all parameters). In contrast, a slight improvement in most parameters, except for concentration, was noted in men with abnormal BSA-volume (+ 0.21 ml), motility (+ 8.72%), and TMC (+ 14.38 M) (p < 0.05 for all parameters). Focusing on men with normal BSA, 33.5% of individuals developed abnormality in one or more of their sperm parameters over time, within a mean time of 1013 ± 661 days. We also found that only time between tests emerged as an independent prognostic factor for the development of abnormal SA later. Interestingly, sperm deterioration in participants in their third, fourth, and fifth decades of life with normal initial semen analysis was similar. Our study provides evidence of a decline in semen quality over time in individuals with normal BSA, in contrast to men with abnormal BSA. Longer time intervals between tests independently increase the risk of sperm abnormalities.

An artificial intelligence algorithm for automated blastocyst morphometric parameters demonstrates a positive association with implantation potential.

Blastocyst selection is primarily based on morphological scoring systems and morphokinetic data. These methods involve subjective grading and time-consuming techniques. Artificial intelligence allows for objective and quick blastocyst selection. In this study, 608 blastocysts were selected for transfer using morphokinetics and Gardner criteria. Retrospectively, morphometric parameters of blastocyst size, inner cell mass (ICM) size, ICM-to-blastocyst size ratio, and ICM shape were automatically measured by a semantic segmentation neural network model. The model was trained on 1506 videos with 102 videos for validation with no overlap between the ICM and trophectoderm models. Univariable logistic analysis found blastocyst size and ICM-to-blastocyst size ratio to be significantly associated with implantation potential. Multivariable regression analysis, adjusted for woman age, found blastocyst size to be significantly associated with implantation potential. The odds of implantation increased by 1.74 for embryos with a blastocyst size greater than the mean (147 ± 19.1 µm). The performance of the algorithm was represented by an area under the curve of 0.70 (p < 0.01). In conclusion, this study supports the association of a large blastocyst size with higher implantation potential and suggests that automatically measured blastocyst morphometrics can be used as a precise, consistent, and time-saving tool for improving blastocyst selection.

Prior exposure to chemotherapy does not reduce the in vitro maturation potential of oocytes obtained from ovarian cortex in cancer patients.

STUDY QUESTION: Does chemotherapy exposure affect IVM potential of immature oocytes retrieved from the ovarian cortex following ovarian tissue cryopreservation (OTC) for fertility preservation? SUMMARY ANSWER: The IVM potential of oocyte retrieved from ovarian cortex following OTC is not affected by prior exposure to chemotherapy but primarily dependent on patient's age, while successful retrieval of immature oocytes from the ovarian tissue is negatively affected by chemotherapy and its timing. WHAT IS KNOWN ALREADY: The potential and feasibility of IVM in premenarche patients was previously demonstrated, in smaller studies. The scarce data that exist on the IVM potential of oocytes retrieved during OTC following chemotherapy support the feasibility of this process, however, this was not previously shown in the premenarche cancer patients population or in larger cohorts. STUDY DESIGN, SIZE, DURATION: A retrospective cohort study evaluating 229 cancer patients aged 1-39 years with attempted retrieval of oocytes from the ovarian tissue and the medium following OTC in a university affiliated fertility preservation unit between 2002 and 2021. PARTICIPANTS/MATERIALS, SETTING, METHODS: A total of 172 chemotherapy naïve and 57 chemotherapy exposed patients aged 1-39 years underwent OTC in university affiliated tertiary infertility and IVF center. OTC and IVM outcomes were compared between the chemotherapy naïve and exposed groups. The main outcome measure was mean IVM rate per patient in the chemotherapy naïve and exposed groups, with subgroup analysis of a 1:1 chemotherapy exposed group matched for age at OTC and type of malignancy. We additionally analyzed premenarche and postmenarche patients' outcomes separately and investigated the effect of time from chemotherapy to IVM, malignancy type and chemotherapy regimen on oocyte number and IVM outcomes in the chemotherapy exposed group. MAIN RESULTS AND THE ROLE OF CHANCE: While the number of retrieved oocytes and percentage of patients with at least one oocyte retrieved was higher in the chemotherapy naïve group (8.7 ± 7.9 versus 4.9 ± 5.6 oocytes and 87.2% versus 73.7%, P < 0.001 and P = 0.016, respectively), IVM rate and number of mature oocytes were comparable between the groups (29.0 ± 25.0% versus 28. 9 ± 29.2% and 2.8 ± 3.1 versus 2.2 ± 2.8, P = 0.979 and P = 0.203, respectively). Similar findings were shown in subgroup analyses for premenarche and postmenarche groups. The only parameter found to be independently associated with IVM rate in a multivariable model was menarche status (F = 8.91, P = 0.004). Logistic regression models similarly showed that past chemotherapy exposure is negatively associated with successful retrieval of oocytes while older age and menarche are predictive of successful IVM. An age and the type of malignancy matched (1:1) chemotherapy naïve and exposed groups were created (25 patients in each group). This comparison demonstrated similar IVM rate (35.4 ± 30.1% versus 31.0 ± 25.2%, P = 0.533) and number of matured oocytes (2.7 ± 3.0. versus 3.0 ± 3.9 oocytes, P = 0.772). Type of malignancy and chemotherapy regimen including alkylating agents were not associated with IVM rate. LIMITATIONS, REASONS FOR CAUTION: This study's inherited retrospective design and the long study period carries the possible technological advancement and differences. The chemotherapy exposed group was relatively small and included different age groups. We could only evaluate the potential of the oocytes to reach metaphase II in vitro but not their fertilization potential or clinical outcomes. WIDER IMPLICATIONS OF THE FINDINGS: IVM is feasible even after chemotherapy broadening the fertility preservation options of cancer patients. The use of IVM for fertility preservation, even after exposure to chemotherapy, should be further studied for optimal postchemotherapy timing safety and for the in vitro matured oocytes potential for fertilization. STUDY FUNDING/COMPETING INTEREST(S): No funding was received for this study by any of the authors. The authors report that no competing interests. TRIAL REGISTRATION NUMBER: N/A.

Delineating the heterogeneity of embryo preimplantation development using automated and accurate morphokinetic annotation.

PURPOSE: Our objective was to design an automated deep learning model that extracts the morphokinetic events of embryos that were recorded by time-lapse incubators. Using automated annotation, we set out to characterize the temporal heterogeneity of preimplantation development across a large number of embryos. METHODS: To perform a retrospective study, we used a dataset of video files of 67,707 embryos from four IVF clinics. A convolutional neural network (CNN) model was trained to assess the developmental states that appear in single frames from 20,253 manually-annotated embryos. Probability-weighted superposition of multiple predicted states was permitted, thus accounting for visual uncertainties. Superimposed embryo states were collapsed onto discrete series of morphokinetic events via monotonic regression of whole-embryo profiles. Unsupervised K-means clustering was applied to define subpopulations of embryos of distinctive morphokinetic profiles. RESULTS: We perform automated assessment of single-frame embryo states with 97% accuracy and demonstrate whole-embryo morphokinetic annotation with R-square 0.994. High quality embryos that had been valid candidates for transfer were clustered into nine subpopulations, as characterized by distinctive developmental dynamics. Retrospective comparative analysis of transfer versus implantation rates reveals differences between embryo clusters as marked by poor synchronization of the third mitotic cell-cleavage cycle. CONCLUSIONS: By demonstrating fully automated, accurate, and standardized morphokinetic annotation of time-lapse embryo recordings from IVF clinics, we provide practical means to overcome current limitations that hinder the implementation of morphokinetic decision-support tools within clinical IVF settings due to inter-observer and intra-observer manual annotation variations and workload constrains. Furthermore, our work provides a platform to address embryo heterogeneity using dimensionality-reduced morphokinetic descriptions of preimplantation development.

Is there a relationship between morphokinetic parameters and neonatal sex in fresh embryo transfers?

To investigate whether morphokinetic parameters differ between male and female embryos in IVF embryos resulting in live births, a retrospective cohort study was undertaken. Files of all live births resulting from a single embryo transfer (SET) cultured in time-lapse incubators between 2013 and 2019 in two tertiary care centres were reviewed. The study group consisted of 187 SETs resulted in 187 live births, of which 100 were females (53.5%) and 87 were males (46.5%). Embryo selection for transfer was based on the known implantation data (KID) score provided by the Embryoscope and morphological assessment by experienced embryologists. Neonatal sex was confirmed through live birth documentation. Morphokinetic parameters and day 3 and day 5 KID scores of male and female embryos were compared. Maternal baseline and treatment characteristics were similar between groups. Morphokinetic time-lapse parameters of male and female embryos including: pronuclei fading; cleavage timings (t2-t9); second and third cell cycle durations; synchrony of the second and third cleavages; late morphokinetic parameters and KID scores did not differ between groups. In conclusion, time-lapse morphokinetic parameters and embryo selection methods do not seem to differ between male and female embryos, and their utilization does not bias towards any neonatal sex.

Dydrogesterone supplementation in addition to routine micronized progesterone administration for luteal support in cycles triggered with lone GnRH agonist results in an acceptable pregnancy rate and avoids the need to freeze embryos.

BACKGROUND: Ovarian hyperstimulation syndrome (OHSS) is reduced when using antagonist cycle with gonadotrophin releasing hormone (GnRH) agonist trigger before ovum pick up. This trigger induces short luteinizing hormone (LH) and follicle stimulating hormone (FSH) peaks, resulting in an inadequate luteal phase and a reduced implantation rate. We assessed whether the luteal phase can be rescued by supplementing with oral dydrogesterone (duphaston) in antagonist cycles after a lone GnRH agonist trigger. METHODS: A retrospective cohort study. The study group (N.=123) included women who underwent IVF. Patients received a GnRH-antagonist with a lone GnRH-agonist trigger due to imminent OHSS. The control group (N.=374) included patients who underwent a standard antagonist protocol with a dual trigger of a GnRH-agonist and human chorionic gonadotrophin (hCG). All the patients were treated with micronized progesterone (utrogestan) for luteal phase support. Study patients were given duphaston in addition. RESULTS: The fertilization rate was comparable between the two groups. The mean number of embryos transferred, the clinical pregnancy rate and the take-home baby rate were comparable between groups (1.5±0.6 vs. 1.5±0.5 and 46.3% vs. 41.2%, and 66.7% vs. 87.7%, respectively). No OHSS event was reported in either group. CONCLUSIONS: This study was the first to evaluate outcomes of duphaston supplementation for luteal support in an antagonist cycle with lone GnRH agonist trigger. The functionality of the luteal phase of those cycles could be restored by adding duphaston. This approach was found to be safe and prevented the need to postpone embryo transfer in case of pending OHSS.

Embryo classification beyond pregnancy: early prediction of first trimester miscarriage using machine learning.

PURPOSE: First trimester miscarriage is a major concern in IVF-ET treatments, accounting for one out of nine clinical pregnancies and for up to one out of three recognized pregnancies. To develop a machine learning classifier for predicting the risk of cleavage-stage embryos to undergo first trimester miscarriage based on time-lapse images of preimplantation development. METHODS: Retrospective study of a 4-year multi-center cohort of 391 women undergoing intra-cytoplasmatic sperm injection (ICSI) and fresh single or double embryo transfers. The study included embryos with positive indication of clinical implantation based on gestational sac visualization either with first trimester miscarriage or live-birth outcome. Miscarriage was determined based on negative fetal heartbeat indication during the first trimester. Data were recorded and obtained in hospital setting and research was performed in university setting. RESULTS: A minimal subset of six non-redundant morphodynamic features were screened that maintained high prediction capacity. Features that account for the distribution of the nucleolus precursor bodies within the small pronucleus and pronuclei dynamics were highly predictive of miscarriage outcome as evaluated using the SHapley Additive exPlanations (SHAP) methodology. Using this feature subset, XGBoost and random forest models were trained following a 100-fold Monte-Carlo cross validation scheme. Miscarriage was predicted with AUC 0.68 to 0.69. CONCLUSION: We report the development of a decision-support tool for identifying the embryos with high risk of miscarriage. Prioritizing embryos for transfer based on their predicted risk of miscarriage in combination with their predicted implantation potential is expected to improve live-birth rates and shorten time-to-pregnancy.

BNT162b2 Covid-19 vaccine does not affect fertility as explored in a pilot study of women undergoing IVF treatment.

BACKGROUND: The COVID-19 pandemic spreads worldwide, causing devastating consequences. BioNTech and Pfizer's BNT162b2 vaccine was one of the first vaccines to receive emergency-use authorization. However, its impact on women's fertility has not been primarily assessed leading to spread of unfounded rumors, causing vaccine hesitancy. We investigate the possible impact of BNT162b2 COVID-19 vaccine on in vitro fertilization (IVF) outcomes. METHODS: We compared data from 42 women undergoing their first IVF following 2 doses of BNT162b2 vaccination to 42 unvaccinated women undergoing their first IVF. The first outcome consisted of the pregnancy based on first hCG value; secondary outcomes were IVF cycle outcomes. A second analysis was done on 29 paired patients from both groups based on age, FSH, and the indication for IVF. RESULTS: We report different levels of the pregnancy formation; oocyte's development: numbers of oocytes retrieved (9.3±6.8 vs. 11.5 ± 7.9, p=0.19) and matured (6.9±4.8 vs. 9.1 ± 6.6, p=0.14), and embryonal stage: fertilization rates (64.1±26.6 vs. 66.3±23.3, p=0.14), the quantity (4.3 ± 3.2 vs. 5.7±4.4, p=0.23) and qualities of embryos (good/fair/poor) at day 3 were comparable. The analysis showed no significant difference between the groups, and the odds for pregnancy were similar (OR=0.9, 95% CL[0.3-2.8] p.value 0.833). CONCLUSIONS: From our results, this vaccine does not affect IVF performance and outcomes from the early stage of oocyte development through to the early beginning of pregnancy; therefore, it seems that the BNT162b2 vaccine does not compromise women's fertility.

Mild COVID-19 Was Not Associated with Impaired IVF Outcomes or Early Pregnancy Loss in IVF Patients.

Data collection regarding the effects of COVID-19 on reproduction is ongoing. This study examined the effect of COVID-19 on IVF cycle parameters and early pregnancy outcomes. It included two arms: the first compared non-exposed cycles to post-SARS-CoV-2 IVF cycles. Sperm parameters were also compared. The second, prospective arm compared pregnancy outcomes among IVF patients who contracted COVID-19 during early pregnancy to those who did not. None of the patients were vaccinated against SARS-CoV-2. The first arm included 60 treatment cycles of women with confirmed COVID-19, compared to 60 non-exposed cycles (either the same patient before exposure or matched non-exposed patients). The outcomes of the treatment cycles did not differ significantly between exposed and non-exposed groups, including number of oocytes, endometrial thickness, fertilization rate and number of top-quality embryos. In 11 cycles, the male partner had also recently recovered: sperm concentration was lower post-exposure: 6.27 million/mL vs. 16.5 pre-exposure (p = 0.008). In 189 patients with IVF-achieved pregnancies, pregnancy loss and hospital admissions did not differ between exposed and non-exposed groups. IVF treatment outcomes and the rate of early pregnancy loss appears to be unaffected by SARS-CoV-2 disease, despite a minor decline in sperm concentration among recent recoverees.

Self-reported elevated pain levels: A coping strategy used by women undergoing medical abortion procedures.

PURPOSE: Epidural analgesia alleviates pain during normal labour but women who undergo medical abortion procedures using epidural analgesia continue to express high pain levels. To understand this we assessed if patients undergoing medical abortions, treated with epidural analgesia, use their pain for psychological benefits. METHODS: This study comprised 105 patients over 13 weeks of gestational age diagnosed with foetal abnormalities after selecting a medical abortion procedure using epidural analgesia. Three questionnaires were handed-out: 1) 'Need for Affect', assessed the motivation to react to emotions; 2) 'Pain-Levels' 3) PANA- 'Positive Affect' (PA), 'Negative Affect' (NA) assessed emotions pre-abortion and post-abortion. RESULTS: Patients with a strong Need for Affect and high Pain-Levels expressed a stronger PA post-abortion (b = .69, se = .11, ß = .68, p < .001, 95%CI [.48,.90]). Patients with a strong NA pre-abortion and high Pain-Levels expressed a higher NA post-abortion (b = .48, se = .11, ß = .53, p < .001, 95%CI [.26, .70]). CONCLUSION: Patients with a strong Need for Affect who express a strong PA pre-abortion intensify their pain to fulfill their Need for Affect, which then helps recovery. Patients with a strong NA pre-abortion and high Pain-Levels indicate a less favourable outcome.

Sperm quality is not affected by the BNT162b2 mRNA SARS-CoV-2 vaccine: results of a 6-14 months follow-up.

PURPOSE: We aimed to investigate the possible effect of SARS-CoV-2 vaccination on sperm quality by evaluating semen analyses of men prior to vaccination and 6-14 months after vaccination. METHODS: This was a retrospective cohort study, conducted in a university-affiliated in vitro fertilization center between October 2021 and March 2022, including men not previously infected with the SARS-CoV-2 virus who received at least 2 doses of the Pfizer-BioNTech (BNT162b2) SARS-CoV-2 vaccine. Semen analyses of samples given pre-vaccination and 6-14 months post-vaccination were analyzed for the parameters of volume, concentration, motility, morphology, and total motile count (TMC) and compared. These parameters were also compared separately for men who received a third (booster) dose and for men with pre-vaccination normal and abnormal sperm. Correlations between time from vaccination and post-vaccination sperm parameters were also assessed. RESULTS: Fifty-eight men were included in the final analysis. Semen volume (2.9 ± 1.4 vs. 2.9 ± 1.6 ml), sperm concentration (42.9 ± 37.9 vs. 51.5 ± 46.2 million/ml), motility (42.5 ± 23.1 vs. 44.3 ± 23.4 percent), morphology (8.8 ± .16.6 vs. 6.6 ± 8.8 percent), and TMC (55.7 ± 57.9 vs. 71.1 ± 77.1 million) were comparable between the pre- and post-vaccination samples. This was true for the entire study cohort, for the subgroup of men who received a third dose and for the subgroups of men with a pre-vaccination normal and abnormal semen samples. No correlation was found between time from vaccination and post-vaccination sperm parameters. CONCLUSIONS: The Pfizer-BioNTech (BNT162b2) SARS-CoV-2 vaccine does not impair any of the sperm parameters over a relatively long-time interval of 6 to 14 months from vaccination.

Pfizer SARS-CoV-2 BNT162b2 mRNA vaccination (BNT162b2) has no adverse effect on elective oocyte cryopreservation outcomes.

RESEARCH QUESTION: Do elective oocyte cryopreservation outcomes in women 1-13 months after SARS-CoV-2 vaccination alter compared with unvaccinated women and do different time intervals between vaccination and ovarian stimulation impact these outcomes? DESIGN: This retrospective cohort study, conducted in a university-affiliated IVF centre, included 232 elective oocyte cryopreservation cycles of vaccinated and unvaccinated patients, without previous infection with the SARS-CoV-2 virus, between December 2020 and January 2022. Two control groups - pre-pandemic (January 2019 to February 2020) and intra-pandemic (December 2020 to January 2022) unvaccinated groups - were compared with the vaccinated group, further divided into four subgroups (under 3, 3-6, 6-9 and 9-13 months). The primary outcome was the elective oocyte cryopreservation cycle outcomes - number of retrieved and number of mature oocytes. RESULTS: The vaccinated group demonstrated comparable outcomes with regards to number of retrieved and mature oocytes compared with the pre-pandemic and intra-pandemic unvaccinated groups (12.6 ± 8.0 versus 13.0 ± 8.2 and 12.5 ± 7.4 retrieved and 10.1 ± 6.9 versus 9.5 ± 6.4 and 10.1 ± 6.3 mature oocytes, respectively; not significant for both). Similar results were noted in a comparison between the intra-pandemic unvaccinated group and the four vaccinated subgroups. No correlation was found between the parameter of days from vaccination and cycle outcomes. Similarly, analysis of covariance showed no association between vaccination status and timing and number of mature oocytes. CONCLUSIONS: The SARS-CoV-2 vaccination does not alter the outcomes of elective oocyte cryopreservation procedures. This is true even in a relatively long time interval of 9 to 13 months from vaccination.

Human blastocyst spontaneous collapse is associated with worse morphological quality and higher degeneration and aneuploidy rates: a comprehensive analysis standardized through artificial intelligence.

STUDY QUESTION: What are the factors associated with human blastocyst spontaneous collapse and the consequences of this event? SUMMARY ANSWER: Approximately 50% of blastocysts collapsed, especially when non-viable, morphologically poor and/or aneuploid. WHAT IS KNOWN ALREADY: Time-lapse microscopy (TLM) is a powerful tool to observe preimplantation development dynamics. Lately, artificial intelligence (AI) has been harnessed to automate and standardize such observations. Here, we adopted AI to comprehensively portray blastocyst spontaneous collapse, namely the phenomenon of reduction in size of the embryo accompanied by efflux of blastocoel fluid and the detachment of the trophectoderm (TE) from the zona pellucida (ZP). Although the underlying causes are unknown, blastocyst spontaneous collapse deserves attention as a possible marker of reduced competence. STUDY DESIGN, SIZE, DURATION: An observational study was carried out, including 2348 TLM videos recorded during preimplantation genetic testing for aneuploidies (PGT-A, n = 720) cycles performed between January 2013 and December 2020. All embryos in the analysis at least reached the time of starting blastulation (tSB), 1943 of them reached full expansion, and were biopsied and then vitrified. PARTICIPANTS/MATERIALS, SETTING, METHODS: ICSI, blastocyst culture, TE biopsy without Day 3 ZP drilling, comprehensive chromosome testing and vitrification were performed. The AI software automatically registered tSB and time of expanding blastocyst (tEB), start and end time of each collapse, time between consecutive collapses, embryo proper area, percentage of shrinkage, embryo:ZP ratio at embryo collapse, time of biopsy (t-biopsy) and related area of the fully (re-)expanded blastocyst before biopsy, time between the last collapse and biopsy. Blastocyst morphological quality was defined according to both Gardner's criteria and an AI-generated implantation score. Euploidy rate per biopsied blastocyst and live birth rate (LBR) per euploid single embryo transfer (SET) were the main outcomes. All significant associations were confirmed through regression analyses. All couple, cycle and embryo main features were also investigated for possible associations with blastocyst spontaneous collapse. MAIN RESULTS AND THE ROLE OF CHANCE: At least one collapsing embryo (either viable or subsequently undergoing degeneration) was recorded in 559 cycles (77.6%) and in 498 cycles (69.2%) if considering only viable blastocysts. The prevalence of blastocyst spontaneous collapse after the tSB, but before the achievement of full expansion, was 50% (N = 1168/2348), irrespective of cycle and/or couple characteristics. Blastocyst degeneration was 13% among non-collapsing embryos, while it was 18%, 20%, 26% and 39% among embryos collapsing once, twice, three times or ≥4 times, respectively. The results showed that 47.3% (N = 918/1943) of the viable blastocysts experienced at least one spontaneous collapse (ranging from 1 up to 9). Although starting from similar tSB, the number of spontaneous collapses was associated with a delay in both tEB and time of biopsy. Of note, the worse the quality of a blastocyst, the more and the longer its spontaneous collapses. Blastocyst spontaneous collapse was significantly associated with lower euploidy rates (47% in non-collapsing and 38%, 32%, 31% and 20% in blastocysts collapsing once, twice, three times or ≥4 times, respectively; multivariate odds ratio 0.78, 95%CI 0.62-0.98, adjusted P = 0.03). The difference in the LBR after euploid vitrified-warmed SET was not significant (46% and 39% in non-collapsing and collapsing blastocysts, respectively). LIMITATIONS, REASONS FOR CAUTION: An association between chromosomal mosaicism and blastocyst collapse cannot be reliably assessed on a single TE biopsy. Gestational and perinatal outcomes were not evaluated. Other culture strategies and media should be tested for their association with blastocyst spontaneous collapse. Future studies with a larger sample size are needed to investigate putative impacts on clinical outcomes after euploid transfers. WIDER IMPLICATIONS OF THE FINDINGS: These results demonstrate the synergistic power of TLM and AI to increase the throughput of embryo preimplantation development observation. They also highlight the transition from compaction to full blastocyst as a delicate morphogenetic process. Blastocyst spontaneous collapse is common and associates with inherently lower competence, but additional data are required to deepen our knowledge on its causes and consequences. STUDY FUNDING/COMPETING INTEREST(S): There is no external funding to report. I.E., A.B.-M., I.H.-V. and B.K. are Fairtility employees. I.E. and B.K. also have stock or stock options of Fairtility. TRIAL REGISTRATION NUMBER: N/A.

How slow is too slow? A comprehensive portrait of Day 7 blastocysts and their clinical value standardized through artificial intelligence.

STUDY QUESTION: What is the clinical value of Day 7 blastocysts? SUMMARY ANSWER: Ending embryo culture at 144 hours post-insemination (h.p.i.; i.e. 6 days) would involve 7.3% and 4.4% relative reductions in the number of patients obtaining euploid blastocysts and live birth(s) (LBs), respectively. WHAT IS KNOWN ALREADY: Many studies showed that Day 7 blastocysts are clinically valuable, although less euploid and less competent than faster-growing embryos. Nevertheless, a large variability exists in: (i) the definition of 'Day 7'; (ii) the criteria to culture embryos to Day 7; (iii) the clinical setting; (iv) the local regulation; and/or (v) the culture strategies and incubators. Here, we aimed to iron out these differences and portray Day 7 blastocysts with the lowest possible risk of bias. To this end, we have also adopted an artificial intelligence (AI)-powered software to automatize developmental timings annotations and standardize embryo morphological assessment. STUDY DESIGN, SIZE AND DURATION: Observational study including 1966 blastocysts obtained from 681 patients cultured in a time-lapse incubator between January 2013 and December 2020 at a private Italian IVF center. PARTICIPANTS/MATERIALS, SETTING, METHODS: According to Italian Law 40/2004, embryos were not selected based on their morphology and culture to ≥168 h.p.i. is standard care at our center. ICSI, continuous culture with Day 5 media refresh, trophectoderm biopsy without assisted hatching and comprehensive chromosome testing (CCT) to diagnose full-chromosome non-mosaic aneuploidies, were all performed. Blastocysts were clustered in six groups based on the time of biopsy in h.p.i. at 12 hr intervals starting from <120 h.p.i. (set as control) up to >168 h.p.i. Blastocyst quality was assessed using Gardner's scheme and confirmed with AI-powered software. AI was also used to automatically annotate the time of expanding blastocyst (tEB) and the hours elapsing between this moment and the achievement of full expansion when blastocysts were biopsied and vitrified. Also, blastocyst area at tEB and at the time of biopsy was automatically assessed, as well as the hour of the working day when the procedure was performed. The main outcomes were the euploidy rate and the LB rate (LBR) per vitrified-warmed euploid single blastocyst transfer. The results were adjusted for confounders through multivariate logistic regressions. To increase their generalizability, the main outcomes were reported also based on a 144-h.p.i. cutoff (i.e. 6 exact days from ICSI). Based on this cutoff, all the main patient outcomes (i.e. number of patients obtaining blastocysts, euploid blastocysts, LBs, with supernumerary blastocysts without a LB and with surplus blastocysts after an LB) were also reported versus the standard care (>168 h.p.i.). All hypothetical relative reductions were calculated. MAIN RESULTS AND THE ROLE OF CHANCE: A total of 14.6% of the blastocysts reached full expansion beyond 144 h.p.i. (5.9% in the range 144-156 h.p.i., 7.9% in the range 156-168 h.p.i. and 0.8% beyond 168 h.p.i.). Slower blastocysts were of a worse quality based on the evaluation of both embryologists and AI. Both later tEB and longer time between tEB and full blastocyst expansion concurred to Day 7 development, quite independently of blastocyst quality. Slower growing blastocysts were slightly larger than faster-growing ones at the time of biopsy, but no difference was reported in the risk of hatching, mainly because two dedicated slots have been set along the working day for these procedures. The lower euploidy rate among Day 7 blastocysts is due to their worse morphology and more advanced oocyte age, rather than to a slower development per se. Conversely, the lower LBR was significant even after adjusting for confounders, with a first relevant decrease for blastocysts biopsied in the range 132-144 h.p.i. (N = 76/208, 36.5% versus N = 114/215, 53.0% in the control, multivariate odds ratio 0.61, 95% CI 0.40-0.92, adjusted-P = 0.02), and a second step for blastocysts biopsied in the range 156-168 h.p.i. (N = 3/21, 14.3%, multivariate odds ratio: 0.24, 95% CI 0.07-0.88, adjusted-P = 0.03). Nevertheless, when the cutoff was set at 144 h.p.i., no significant difference was reported. In this patient population, ending embryo culture at 144 h.p.i. would have caused 10.6%, 7.3%, 4.4%, 13.7% and 5.2% relative reductions in the number of patients obtaining blastocysts, euploid blastocysts, LBs, supernumerary blastocysts without an LB and surplus blastocysts after an LB, respectively. LIMITATIONS, REASONS FOR CAUTION: Gestational and perinatal outcomes were not assessed, and a cost-effectiveness analysis is missing. Moreover, we encourage other groups to investigate this topic with different culture and biopsy protocols, as well as in different clinical settings and regulatory contexts. WIDER IMPLICATIONS OF THE FINDINGS: In view of the increasing personalization and patient-centeredness of IVF, whenever allowed from the local regulations, the choice to culture embryos to Day 7 should be grounded on the careful evaluation of couples' reproductive history. Patients should be aware that Day 7 blastocysts are less competent than faster-growing ones; still, poor prognosis couples, couples less compliant toward other attempts in case of a failure and couples wishing for more than one child, may benefit from them. AI tools can help improving the generalizability of the evidence worldwide. STUDY FUNDING/COMPETING INTEREST(S): This study did not receive any funding. I.E., A.B.M. and I.H.-V. are employees of Fairtility Ltd. TRIAL REGISTRATION NUMBER: N/A.

In silico Docking Analysis for Blocking JUNO-IZUMO1 Interaction Identifies Two Small Molecules that Block in vitro Fertilization.

Combined hormone drugs are the basis for orally administered contraception. However, they are associated with severe side effects that are even more impactful for women in developing countries, where resources are limited. The risk of side effects may be reduced by non-hormonal small molecules which specifically target proteins involved in fertilization. In this study, we present a virtual docking experiment directed to discover molecules that target the crucial fertilization interactions of JUNO (oocyte) and IZUMO1 (sperm). We docked 913,000 molecules to two crystal structures of JUNO and ranked them on the basis of energy-related criteria. Of the 32 tested candidates, two molecules (i.e., Z786028994 and Z1290281203) demonstrated fertilization inhibitory effect in both an in vitro fertilization (IVF) assay in mice and an in vitro penetration of human sperm into hamster oocytes. Despite this clear effect on fertilization, these two molecules did not show JUNO-IZUMO1 interaction blocking activity as assessed by AVidity-based EXtracellular Interaction Screening (AVEXIS). Therefore, further research is required to determine the mechanism of action of these two fertilization inhibitors.

The BNT162b2 mRNA Covid-19 vaccine does not impair sperm parameters.

RESEARCH QUESTION: Does the BNT162b2 COVID-19 vaccine affect sperm parameters of patients with a normal or an abnormal semen analysis? DESIGN: Data were collected from male patients undergoing IVF treatment after completing vaccination between February 2021 and June 2021 (post-vaccine). For comparison, records of the same patients were reviewed before the vaccination (pre-vaccine) back to January 2017. Patients with azoospermia were excluded. Sperm parameters were compared between pre- and post-vaccine groups. Each patient served as self-control. RESULTS: Seventy-two patients were included in the study (median interquartile range [IQR] age 35.7 [33.0-43.0] years), of whom 57 had a normal semen analysis. The time between the first vaccine and the post-vaccine sperm analysis was 71.0 (40.5-104.8) days. The sperm parameters before and after the vaccination were as follows: sperm volume before 3.0 (2.0-4.0) and after 3.0 (1.6-3.9) ml, P = 0.02; sperm concentration before 26.5 (14.0-64.7) and after 31.0 (14.2-80.0) 106/ml, P = 0.35; and total motile sperm count before 33.7 (9.0-66.0) and after 29 (6.0-97.5)106, P = 0.96. Sub-group analyses were conducted for patients with male infertility and patients with a normal semen analysis. Neither of the sub-groups showed significant changes after vaccination. CONCLUSION: Sperm parameters showed no significant changes after vaccination among men with a normal and abnormal semen analysis. Therefore, the BNT162b2 vaccine does not seem to affect sperm parameters. The preliminary results are reassuring for the entire global population, currently undergoing intense vaccination campaigns against COVID-19.

Obstetric outcomes of young women following in-vitro fertilization: a case-control study.

BACKGROUND: Women undergoing in-vitro fertilization (IVF) treatments are at increased risk for maternal and neonatal complications compared to women who conceive spontaneously. Though spontaneous pregnancies of young women and adolescents have an increased risk for adverse maternal and neonatal outcomes, pregnancy outcomes of this age group, following IVF treatment have been scarcely reported. The aim of this study was to report maternal and neonatal outcomes of young women who conceived following IVF compared to women in the same age group with spontaneous conception. METHODS: We performed a multicenter case-control study. The study group included women aged 17-25 years who conceived by IVF with an ongoing singleton pregnancy. For the purpose of the study, a control group matched (1:2 ratio) for maternal age at delivery and parity was constructed. Demographic, medical history, pregnancy related characteristics and maternal and neonatal outcomes were compared between groups. Finally, factors associated with spontaneous vaginal delivery were assessed for the entire cohort using a univariate and multivariate logistic regression model. RESULTS: Between 2005 and 2021, we identified 80 women aged 19-25 years who conceived by IVF. A control group of 160 women was matched to the study group by age and parity. The unmatched maternal characteristics and pregnancy associated complications were similar among the groups. However, the IVF group had a significantly higher rate of induction of labor (48.1% vs. 26.6%, p = 0.001), meconium-stained amniotic fluid (27.6% vs. 14.1%, p = 0.025), prolonged second stage of labor (26.0% vs. 7.3%, p = 0.001) and operative vaginal delivery (22.5% vs.12.5%, p = 0.048). Neonatal outcomes were for the most part comparable; nevertheless, we found a higher rate of neonates with an umbilical artery pH < 7.1 in the IVF group (9.8% vs. 0.0%, respectively; p = 0.022). A logistic regression analysis for spontaneous vaginal delivery (vs. cesarean or operative vaginal deliveries) found that spontaneous onset of labor (vs. induction of labor) (OR = 2.08; 95% CI = 1.07-4.05, p = 0.03) was positively associated with spontaneous vaginal delivery while prolonged second stage of labor (OR = 0.35; 95% CI = 0.13-0.95, p = 0.04) was negatively associated with this parameter. CONCLUSION: Young women who conceive by in-vitro fertilization are expected to reach favorable pregnancy outcomes, comparable to women who conceived spontaneously.

Do vitrified frozen-thawed embryos generated from successful fresh cycles have higher potential for favorable outcome?

BACKGROUND: Many variables, including woman's age, embryo quality and endometrial receptivity, influence the success rate of either fresh embryo transfer (ET) or frozen-thawed ET (FT-ET) cycles. This study aims to examine whether the results of a fresh in-vitro fertilization (IVF) cycle correlate with its frozen-thawed embryos transfer outcomes. METHODS: A retrospective cohort study conducted in a tertiary medical center single IVF unit between 2014-2017. The study population was comprised of women who underwent fresh ET with the remaining embryos frozen using vitrification and at least one consecutive FT-ET cycle. Comparison of FT-ET cycle's details and outcomes were assessed in relation to the outcome of preceding fresh ET cycle. RESULTS: A total of 599 women underwent fresh ET and at least one consecutive FT-ET cycle. Significantly higher rates of successful FT-ET cycle outcomes were observed in the group of patients with a successful fresh cycle compared to the group with an unsuccessful fresh cycle (48.9% vs. 20.8%, P<0.001). Logistic regression demonstrated an adjusted OR of 5.02 for successful FT-ET after a successful fresh cycle. CONCLUSIONS: Frozen-thawed embryos generated from successful fresh IVF cycles have higher potential to implant when compared to frozen-thawed embryos obtained in an unsuccessful fresh cycle.

High ovarian response to ovarian stimulation: effect on morphokinetic milestones and cycle outcomes.

PURPOSE: To assess the effect of high ovarian response on oocyte quality and ovarian stimulation cycle outcomes. METHODS: A retrospective cohort study conducted at three IVF units. The high ovarian response (HOR) and polycystic ovary syndrome (PCOS) with HOR (PCOS HOR) groups included 151 and 13 women who underwent controlled ovarian stimulation (COS) resulting in more than 15 retrieved oocytes, for a total of 1863 and 116 cultured embryos, respectively. The normal ovarian response (NOR) group comprised 741 women with 6-15 retrieved oocytes, resulting in 4907 cultured embryos. Data collected included fresh cycle data and pregnancy rates, in addition to annotation of morphokinetic events from time of pronuclei fading to time of initiation of blastocyst formation of embryos cultured in a time lapse incubator, including occurrence of direct unequal cleavage at first cleavage (DUC-1) (less than 5 h from two to three blastomeres). Comparison was made between morphokinetic parameters between the 3 groups. Cycle outcomes were compared in the high vs. normal ovarian response groups. RESULTS: Oocyte maturation rate was significantly lower in the HOR vs. NOR groups (56.5% vs. 90.0%, p < 0.001), while the fertilization rates were similar (60.2% vs. 58.1%, p = 0.397). The prevalence of DUC-1 embryos was higher in the PCOS HOR and the HOR groups as compared to the NOR group (22.7% vs. 16.2% and 12.0%, respectively, p < 0.001). After exclusion of DUC-1 embryos, remaining embryos from the NOR and HOR groups reached the morphokinetic milestones at similar rates, with comparable implantation and clinical pregnancy rates, while the PCOS HOR showed shorter time to 5 blastomeres compared to the NOR and HOR groups. CONCLUSIONS: High ovarian response might be associated with decreased oocyte quality, manifested as a higher proportion of immature oocytes and higher rate of direct uneven cleavage embryos, while embryos exhibiting normal first cleavage have similar temporal milestones and implantation potential.

Endometrial thickness following early miscarriage in IVF patients - is there a preferred management approach?

BACKGROUND: Endometrial thickness (ET) has previously been shown to positively correlate with implantation and clinical pregnancy rates. Pregnancies achieved using in-vitro fertilization (IVF) technique are prone to higher rates of early miscarriage. The aim of this study was to compare the effects of expectant management, medical treatment (Misoprostol) and dilation and curettage (D&C) for early miscarriage following IVF cycles on the subsequent cycle outcomes - endometrial thickness and reproductive outcomes. METHODS: A retrospective cohort study of women who underwent embryo transfer, conceived and had first trimester miscarriage with at least one subsequent embryo transfer. ET measurements during fresh or frozen-thawed IVF cycles were assessed for each patient. Comparisons of ET differences between the miscarriage and the subsequent cycles, as well as reproductive outcomes, were performed according to the initial miscarriage management approach. RESULTS: A total of 223 women were included in the study. Seventy-eight women were managed conservatively, 61 were treated with Misoprostol and 84 women underwent D&C. Management by D&C, compared to conservative management and Misoprostol treatment was associated with higher prevalence of a significant (> 2 mm) ET decrease (29.8%% vs. 14.1and 6.6%, respectively; p < .001) and was the only approach associated with a significant increase in the rates of ET under 7 and 8 mm in the following cycle (p = 0.006 and 0.035; respectively). Clinical pregnancy rates were significantly lower following D&C compared with conservative management and Misoprostol (16.7% vs. 38.5 and 27.9%, respectively; p = 0.008) as well as implantation rate (11.1% vs. 30.5.% and 17.7, respectively; p < 0.001). CONCLUSION: Our data suggest that D&C management of a miscarriage is associated with decreased ET and higher rates of thin endometrium in the subsequent IVF cycle, compared with conservative management and Misoprostol treatment. In addition, implantation and pregnancy rates were significantly lower after D&C.