Study question How demanding in terms of time and resource allocation is the full integration of an AI platform to the routine operation of an IVF clinic? Summary answer The rapid and effective implementation, and continuous performance, of EMA was qualitatively and quantitatively demonstrated for the first time in a real-world clinical setting. What is known already The role of AI-based embryo predictive analysis tools is often hailed as one of the most important recent developments in the IVF clinic. The high precision of such systems has been reported. For instance, the AI-system implemented here, EMATM(AiVF), employs a convolutional neural network architecture, providing an area-under-the-curve (AUC) of 0.95 with 83% accuracy. However, little was reported to date on the full implementation of these advanced systems in the active IVF lab. This study prospectively evaluated the clinical implementation and process-of-use in an IVF clinic of EMA, the first end-to-end AI-driven platform designed to algorithmically aid in evaluating embryos. Study design, size, duration A prospective observational single center study. The study was performed in two phases. Phase I: EMA was integrated into standard workflow and qualitatively evaluated over the course of one month by five embryologists in a series of twice-daily qualitative checks and questionaries. Phase II: The rate of agreement between EMA and embryologists were benchmarked and compared to evaluate how the model aids embryologists in efficiently assessing embryos. Participants/materials, setting, methods Phase I: Five senior embryologists completed electronic questionaries to qualitatively report on the ease-of-use, functionality, and performance of EMA after using the platform as adjunctive information on 588 embryos; ICSI was performed on all treatment cycles. Phase I was completed within 2 weeks. Phase II: The rate of agreement between five senior embryologists and EMA was calculated for the accuracy in ranking embryo(s) for transfer/freeze (146 treatment cycles). Phase II was undertaken in 4 weeks. Main results and the role of chance EMA was effectively incorporated into a busy IVF laboratory for routine daily use to algorithmically assess all embryos at 105 hours post-fertilization prior to vitrification. All embryos were cultured in a time-lapse incubator and successfully evaluated by both EMA and embryologists in parallel to conventional morphologic embryo evaluation. In Phase I, all five embryologists qualitatively approved of EMA’s integration and clinical utility inside their workflow and reported enhanced efficiency when EMA was used per its intended use. In Phase II we demonstrated a 86% agreement rate between embryologists and EMA. Of all embryos selected for transfer by embryologists 100% were also identified by EMA as having the highest potential for implantation. Of all embryos selected for vitrification by embryologists, 85%were identified by EMA as top-quality (Gardner criteria: A/B grade) embryos. Among embryos that were graded C/D (Gardner criteria) by embryologists, 89% were identified by EMA as “low grade” as well. Pregnancies were shown to be highly associated with EMA's embryo selection. The final stage of our implementation analysis of EMA is currently ongoing; the association between the algorithmic outputs of EMA and clinical implantation rates are being investigated in a prospective double-blinded, observation cohort study and results will be presented. Limitations, reasons for caution This is a single center study, based on relatively homogenous patient population. Nevertheless, given the impressive results reported herein, we conclude that this single case-study is sufficient for demonstrating rapid and successful implementation and process-validation of EMA for routine use by the clinic. Wider implications of the findings AI-based decision support systems like EMA have the potential to increase rapid and objective standardization inside the clinic, thereby improving accurate decision making and saving time and resources without interfering with the busy workflow of an IVF setting. Routine use of EMA in IVF should be prioritized for further evaluation. Trial registration number N/A

Study question Are there differentially expressed genes (DEGs), in the testicular tissue of azoospermic males which could uncover tissue specific gene expression signatures, associated with idiopathic azoospermia? Summary answer The current findings link for the first time the pathophysiology of idiopathic azoospermia to the immune system and open a new era for further investigation What is known already Azoospermia is the most severe form of male infertility and affects 10% of the infertile men and 1% of the general male population which equals to several million men all over the world. A causal genetic factor is established in only 25% of the cases while in the remaining 75% there is no diagnosis and is termed idiopathic. Published data demonstrate that in idiopathic azoospermia heterogeneous genetic factors are the underlying cause. Clarifying the genetic basis of azoospermia will immensely improve our current clinical and treatment approaches for patient care at the levels of diagnosis, therapy, treatment, and safety Study design, size, duration During June 2020 and June 2021 testicular samples from 26 consented IVF patients were included in this study. The samples were divided in 7 pools based on the presence of testicular spermatozoa found. In particular, Pools 1-3 included testicular samples with high, average and very low presence of spermatozoa, Pools 4 & 5 samples with no spermatozoa found, and Pools 6-7 included only CF carriers. Pools 6 &7 were used as controls. Participants/materials, setting, methods Total RNA was isolated using the RΝeasy plus universal mini kit (Qiagen) and quantified using a photometer. Next, RNA sequencing was performed by Novogene, using the Illumina NovaSeq platform and the DEGs that were identified were processed using Gene Ontology (GO) functional enrichment analysis, pathway enrichment analysis and protein-protein interaction (PPI) network reconstruction using the Gene Ontology Resource, the KEGG database and the STRING database respectively. Main results and the role of chance In total RNA from up to 39.664 genes was detected and significant differences in gene expression (DEGs, log2fold change≥2, p-value < 0.05) were identified in the 7 pools analyzed. The top 10% of the protein coding DEGs from each comparison was further analyzed. Up to 1.900 protein coding genes were found to be common in at least two comparisons and exhibit consistent expression profiles. In particular, 920 genes were overexpressed in the “good quality” testicular samples (high & average presence of spermatozoa) while 940 genes were overexpressed in the “low quality” testicular samples (rare or no spermatozoa found). GO analysis revealed that the sexual reproduction, male gamete generation spermatogenesis, cilium movement and fertilization biological processes were enriched in the “good quality” testicular pools while the biological processes response to stimulus, response to stress, response to cytokine and defense response were overrepresented in the “low quality” testicular samples. The PPI network of the DEGs was reconstructed and hub proteins with a putative key role in the pathogenesis of azoospermia were identified. Limitations, reasons for caution While transcriptomics analysis is becoming a powerful analysis tool there are still technical limitations which may impact the accurate representation of the DEGs profiles found. The data obtained in this study represent the whole testicular tissue samples analyzed and they are not cell-type specific Wider implications of the findings The findings of this study demonstrate that in the “low quality” testicular samples, there is a significant under-expression of testis-specific genes directly involved in spermatogenesis and fertilization processes, an over-expression of the genes involved in the body’s immune defense system and advance our understanding in the pathophysiology of idiopathic azoospermia. Trial registration number Τ1EDK-02787

Study question Does laser assisted hatching (ΑΗΑ) of vitrified-warmed blastocysts post warming improve the pregnancy rates? Summary answer This study suggests that although laser assisted hatching post warming shows a trend towards improving pregnancy rates, the difference is not significant. What is known already It is known that vitrification may alter the zona pellucida’s biochemical properties and possibly cause hatching failure and ultimately implantation failure. One proposed way in order to overcome this drawback, is the implementation of laser assisted hatching. During assisted hatching, multiple laser shots are performed in the perivitelline space creating an opening on the zona pellucida which facilitates the blastocyst’s herniation process. The efficiency of this method in regard to pregnancy outcomes though, remains controversial. Some studies report a significantly positive effect on pregnancy outcomes using laser assisted hatching whereas others report no significant difference. Study design, size, duration This prospective randomized study was performed at Embryolab Fertility Clinic, in Thessaloniki, Greece between January 2020 and October 2020 and included 2439 frozen embryo transfers. Patients with vitrified-warmed embryos were randomized and allocated to the study (ΑHA) group or control (NO AHA) group. Participants/materials, setting, methods Patients were divided in two groups: AΗΑ group (n = 1799) where laser assisted hatching was performed on the day of embryotransfer (Day 3 or Day 5) on the zona of the embryos post warming and control group (n = 640) where the embryos remained untreated after the warming procedure. The 2 groups were further divided in 3 subgroups depending on the women’s’ age (≤35, 36-40, ≥41) and in 2 more subgroups depending on the day of the transfer. Main results and the role of chance Mean pregnancy rate for all embryos’ stages in AHA group was 60.03% whereas in the NO AHA group was 58.28% (p = 0.4385). In the subgroup of ≤ 35-years, pregnancy rates were 70.23% and 66.49% respectively (p = 0.3363). In the 36-40years subgroup, rates were 58.24% and 60.09% respectively (p = 0.6418). And finally, in the ≥41-years subgroup, pregnancy rates were 54.26% and 49.79% respectively (p = 0.2331). In the group where the embryos were on cleavage stage, the overall pregnancy rate for the AHA group was 27.46 and for the NO AHA group was 35.42% (p = 0.2957). In the subgroup of ≤ 35-years, pregnancy rates were 44.44% and 58.33% respectively (p = 0.4231). In the 36-40years subgroup, rates were 33.33% and 42.86% respectively (p = 0.5236). And finally, in the ≥41-years subgroup, pregnancy rates were 18.42% and 18.18% respectively (p = 0.9796). In the group where the embryos were on blastocyst stage, the overall pregnancy rate for the AHA group was 62.82% and for the NO AHA group was 60.14% (p = 0.2486). In the subgroup of ≤ 35-years, pregnancy rates were 71.66% and 67.03% respectively (p = 0.2430). In the 36-40years subgroup, rates were 60.16% and 61.31% respectively (p = 0.7786). And finally, in the ≥41-years subgroup, pregnancy rates were 58.37% and 53.08% respectively (p = 0.1763). Limitations, reasons for caution Clinical pregnancy rates and live birth rates were not available so as to draw a safer conclusion regarding the effectiveness of the method. In contrast to all kind of cases (both homologous and heterologous cycles) having been pooled together, an appropriate subgrouping might would have been the optimal approach. Wider implications of the findings AHA before transfer of vitrified/warmed embryos does not improve pregnancy rates regardless of stage or age. Nevertheless of the trend towards improving pregnancy rates, the difference is not statistically significant. Large scale, well-designed and appropriately subgrouped studies are necessary in order to investigate if this trend can become statistically significant. Trial registration number N/A

Study question Does vitrification of oocytes in oocyte donation program alter the morphokinetic pattern of human embryos? Summary answer Both the extrusion of the second polar body (tPB2) and the appearance of the pronuclei (tPNa) occur earlier in vitrified oocytes without affecting further development. What is known already Vitrification of oocytes has been a breakthrough in oocyte cryopreservation and significantly altered practices in IVF labs. Oocyte vitrification/warming has become an integral part of daily routine and thorough assessment of its efficiency is crucial. Though, studies of vitrified-warmed oocytes have shown similar fertilization, cleavage, blastulation, pregnancy and live birth rates to fresh oocytes, the morphokinetic pattern of embryos originating from vitrified oocytes has not been thoroughly studied. According to some studies vitrification may be related to delayed blastocyst formation while others failed to see any alteration in the morphokinetic pattern between embryos derived from fresh or frozen oocytes. Study design, size, duration This retrospective observational study was performed at Embryolab Fertility Clinic, in Thessaloniki, Greece between April 2020 and September 2021 and included 590 vitrified oocytes from 68 oocyte donation cycles. Control group consisted of 31 good prognosis cases (oocyte donation cycles or homologous oocyte cycles of women under 35 years old). Participants/materials, setting, methods 68 vitrified oocyte donation cycles and 31 fresh control cycles were analyzed. All oocytes after ICSI were cultured in Embryoscope time-lapse incubator up to blastocyst stage. Key time parameters and dynamic events were analyzed using generalized estimating equations (GEE) regression analysis for the non-independent nature of the data. Moreover, the following comparisons were performed between groups: fertilization, cleavage, top cleavage, blastocyst, top blastocyst and pregnancy rates. Main results and the role of chance There was no significant difference in fertilization rates between control and frozen oocytes (77.79% ± 15.73 vs 71.26% ± 21.50 respectively, p = 0.15). Cleavage rate and top cleavage rate (more than 6 cells, equal size, with no or minor fragmentation), were higher in frozen oocytes with this difference being significant (p = 0.02 for cleavage rate and p = 0.015 for top cleavage rate). Blastocyst rate and top blastocyst (2AA, 3AA, 3AB, 4AA, 4AB according to Gardner criteria) rate, though, showed similar rates with no statistically significant difference (Blastocyst rate: 74,15% ± 17.19-control vs 76.42% ± 30.75-frozen oocytes, p = 0.6 & Top blastocyst rate: 45.54% ± 24.04-control, 45.30% ± 32.04-frozen oocytes, p = 0.82). Pregnancy rates were not significantly different (control:85.19%, frozen oocytes:75.38%, p = 0.29). The comparison for key time parameters and dynamic events that were analyzed (tPB2, tPNa, tPNf, t2, t3,t4, t5, t6, t7, t8, t9+, tSC, tM, tSB, tB, tEB, S2, S3, S5, CC1, CC2, CC3,Compaction and Blastulation) showed significant difference in 2 time parameters: tPB2 (4.62 hours-control, 3.67 hours-frozen, Coef: -1.09, p = 0.015) and tPNa (9.07 hours-control, 8.25 hours-frozen oocytes, Coef: -1.06, p = 0.043). Limitations, reasons for caution The fact that the oocytes under comparison were not sibling as well as its retrospective nature, constitute the main limitations of the present study. Moreover, the number of cases included in this study was limited and the available information about ongoing pregnancy and live births is still pending. Wider implications of the findings tPB2 and tPNa seem to occur earlier in vitrified oocytes without affecting further development, implying a need for reviewing timings between OPU/vitrification/warming/ICSI. Moreover, vitrified oocytes displayed higher cleavage and top cleavage rates with unaffected blastocyst or pregnancy rates. Overall, vitrification of oocytes seems to be safe and reliable. Trial registration number N/A