Ettie Maman

Purpose: Evaluate outcomes of Day-4 embryo transfer according to embryonic developmental stage. Methods: This retrospective single-center study included fresh IVF/ICSI cycles with day-4 single embryo transfer between January 2021 and July 2025. Cycles with double embryo transfer, rescue ICSI, or missing follow-up were excluded. Transfers were divided into three groups based on embryonic stage at transfer: cleavage, morula or blastocyst-stage. Demographic and cycle characteristics, as well as clinical outcomes, were compared. Clinical pregnancy was the primary outcome. Results: A total of 669 day-4 single fresh embryo transfer cycles were included: 130 cleavage-stage (19.4%), 449 morula-stage (67.1%), and 90 blastocyst-stage (13.5%). Compared to the morula stage, the cleavage-stage group was characterized by an older age, lower oocyte yield and fewer 2PN embryos, while the blastocyst-stage group was characterized by a younger age and a similar oocyte yield and 2PN embryos. Clinical pregnancy rates increased significantly with advancing embryonic stage (10.0%, 32.96%, and 48.89%, respectively; p < 0.05). In multivariable analysis, only increasing maternal age (OR 0.92, 95% CI 0.88–0.96) and cleavage-stage transfer (OR 0.37, 95% CI 0.18–0.78) remained negatively associated with clinical pregnancy. Conclusions: Fresh day 4 embryo transfer outcomes are strongly influenced by embryonic developmental stage. Cleavage-stage embryos on day 4 are associated with poor pregnancy rates and may benefit from extended culture. In contrast, day 4 morula or blastocyst transfer represents a viable clinical option, particularly when logistical constraints preclude day 5 transfer.

STUDY QUESTION: Can vitrification with a shortened equilibration solution (ES) be safely applied to human embryos without compromising key embryological clinical outcomes? SUMMARY ANSWER: Vitrification with shortened equilibration was associated with comparable observed survival, developmental progression, and blastocyst re-expansion rates when compared to standard vitrification. WHAT IS KNOWN ALREADY: Vitrification with shortened-equilibration exposure has been primarily evaluated in mouse models and in human oocytes at the germinal vesicle and metaphase II stages, where short exposure times have been shown to maintain post-warming survival and preserve mitochondrial integrity comparable to conventional vitrification. The European Commission (CE) approval exists for the use of shortened-equilibration vitrification protocols in human oocytes, although clinical experience remains limited. Conversely, evidence regarding the application of shortened-equilibration vitrification to human embryos is currently lacking, and there is currently no clinical regulatory approval for embryos or blastocyst vitrification using shortened-equilibration protocols. STUDY DESIGN, SIZE, DURATION: Prospective proof-of-concept study performed in a single IVF laboratory from April 2025 to March 2026. It involved 45 human blastocysts, 49 cleavage-stage embryos, and 102 tripronuclear (3PN) zygotes. Blastocysts were allocated to standard, shortened-equilibration vitrification using full-volume (300 µl) or microdrop loading (50 µl) vitrification. 3PN zygotes were used as a model for early-stage embryo response and were vitrified immediately after fertilization or at the cleavage stage according to group allocation, using standard and shortened-equilibration protocols. PARTICIPANTS/MATERIALS, SETTING, METHODS: All blastocysts were identified as affected by preimplantation genetic testing for monogenic disorders (PGT-M) and used for research only; they were cryopreserved, then warmed using standard protocols, then re-vitrified per group using shortened-equilibration vitrification and subsequently warmed using one-step warming. 3PN zygotes were included as clinically relevant research material and assigned to standard vitrification or shortened-equilibration vitrification (300 µl, 2 min; microdrops 50 µl, 2–3 min). Outcomes included survival rate, day-3 development, blastocyst formation, iDAScore (morphokinetic assessment), and post-warming re-expansion. MAIN RESULTS AND THE ROLE OF CHANCE: 3PN zygotes were allocated to four groups: standard vitrification and shortened-equilibration vitrification using full-volume loading (300 µl, 2-min) or microdrop loading (50 µl, 2 or 3-min). Baseline characteristics were comparable between groups. Post-warming survival rates ranged from 89.2% to 100% (P = 0.408). Day-3 development ranged from 47% to 60% (P = 0.868). Blastocyst formation ranged from 32.0% to 41.2% (P = 0.920). The mean iDAScore ranged from 2.25 to 4.07 (P = 0.281). In cleavage-stage embryos, survival ranged from 78.6% to 95.2% (P = 0.325), and blastocyst formation from 25.0% to 55% (P = 0.262). In blastocysts, survival ranged from 93.3% to 100% (P = 0.592). Re-expansion at 2 h ranged from 82% to 90% after first warming (P = 0.156) and from 82% to 85% after second warming (P = 0.784). Time to complete re-expansion ranged from 3.6 to 4.0 h (P = 0.893). LIMITATIONS, REASONS FOR CAUTION: The sample sizes were limited, and the study may be underpowered to detect differences between groups; patient distribution was unbalanced due to random allocation of samples between groups. In addition, the use of 3PN zygotes limits direct comparison with established blastocyst quality benchmarks due to their inherently reduced developmental potential. WIDER IMPLICATIONS OF THE FINDINGS: Shortened-equilibration vitrification demonstrated comparable post-warming survival, developmental progression, and blastocyst re-expansion to standard vitrification. These findings support the feasibility of shortening equilibration time without compromising key embryological outcomes. If validated in larger studies, this approach may contribute to optimizing and simplifying vitrification protocols in clinical practice. FUNDING: This research received no specific external grant from funding agencies in the public, commercial, or not-for-profit sectors. Departmental and institutional resources from the Infertility and IVF Institute, Sheba Medical Center, supported the study and manuscript preparation. The laboratory also receives general research scholarship support from the Alrov Fund. DISCLOSURES: R.O. reports speaker's fees from Merck Group. The other authors declare that they have no competing interests. TRIAL REGISTRATION NUMBER: N/A

Our aim was to develop an AI-based NC-FET Treatment Management Algorithm (NTMA), decision-support system that predicts ovulation in real time to manage and optimize natural frozen embryo transfer cycle (NC-FET) scheduling. The algorithm was developed using a “teacher-student” machine learning approach and was trained on a total of 3,975 labeled NC-FET, including 3,432 training cycles and 543 test cycles. A second test group included 166 documented ovulation cycles (documented follicular rupture and LH surge in two consecutive days of ultrasound scans). The algorithm showed high ovulation detection accuracy in both tests’ groups particularly one day before and the day of ovulation (95.4% and 94.6% in the labeled test group and 95.5% and 95.3% in the documented test group, respectively). Most influential predictive features of the algorithm included LH levels, the estrogen/progesterone ratio, and leading follicle size during the monitored test days. The NTMA yielded 92.04% correct prediction 7 in identifying ovulation with an average of 3.1 tests per cycle. We propose an AI-based algorithm for the complete management of a NC-FET. The algorithm shows high accuracy in predicting the time of ovulation and therefore can serve as a useful decision support tool for clinicians in their daily practice. Prospective studies are warranted to validate these results.

This study aims to develop physician support software for determining ovulation time and assess its impact on pregnancy outcomes in natural cycle frozen embryo transfers (NC-FET). To develop, assess, and validate an ovulation prediction model, three datasets were used: REI Ovulation Determination dataset (500 cycles) split into training (309), validation (90), and test (101) sets; the Documented Ovulation dataset (101 cycles) with confirmed ovulation (documented follicular rupture and LH surge); and the Clinical Pregnancy Rates dataset (515 NC-FET cycles), categorized into “Matched” and “Mismatched” based on alignment with the model’s ovulation determination. Pregnancy outcomes were compared between the groups. The ovulation prediction model exhibited 93.85% and 92.89% matching rates with the REI Ovulation Determination and Documented Ovulation datasets, respectively. In the Clinical Pregnancy Rates dataset, the Matched group (282 cycles) showed significantly higher clinical pregnancy rates than the Mismatched group (34.6% vs. 25.9%, p = 0.04) and similar results for patients under 37 (41.1% vs. 30.7%, p = 0.04). Logistic regression indicated lower pregnancy rates in Mismatched cases (odds ratio 0.67 for the general population, 0.63 for patients under 37). In conclusion, we introduce a highly accurate AI ovulation prediction model. Treatment cycles aligning with the model’s recommendations had significantly increased clinical pregnancy rates.

Ultrasound monitoring and hormonal blood testing are considered by many as an accurate method to predict ovulation time. However, uniform and validated algorithms for predicting ovulation have yet to be defined. Daily hormonal tests and transvaginal ultrasounds were recorded to develop an algorithm for ovulation prediction. The rupture of the leading ovarian follicle was a marker for ovulation day. The model was validated retrospectively on natural cycles frozen embryo transfer cycles with documented ovulation. Circulating levels of LH or its relative variation failed, by themselves, to reliably predict ovulation. Any decrease in estrogen was 100% associated with ovulation emergence the same day or the next day. Progesterone levels > 2 nmol/L had low specificity to predict ovulation the next day (62.7%), yet its sensitivity was high (91.5%). A model for ovulation prediction, combining the three hormone levels and ultrasound was created with an accuracy of 95% to 100% depending on the combination of the hormone levels. Model validation showed correct ovulation prediction in 97% of these cycles. We present an accurate ovulation prediction algorithm. The algorithm is simple and user-friendly so both reproductive endocrinologists and general practitioners can use it to benefit their patients.

The role of prostaglandins (PGs) in the ovulatory process is known. However, the role of the ATP binding cassette subfamily C member 4 (ABCC4), transmembrane PG carrier protein, in ovulation remains unknown. We report herein that ABCC4 expression is significantly upregulated in preovulatory human granulosa cells (GCs). We found that PGE2 efflux in cultured human GCs is mediated by ABCC4 thus regulating its extracellular concentration. The ABCC4 inhibitor probenecid demonstrated effective blocking of ovulation and affects key ovulatory genes in female mice in vivo. We postulate that the reduction in PGE2 efflux caused by the inhibition of ABCC4 activity in GCs decreases the extracellular concentration of PGE2 and its ovulatory effect. Treatment of female mice with low dose of probenecid as well as with the PTGS inhibitor indomethacin or Meloxicam synergistically blocks ovulation. These results support the hypothesis that ABCC4 has an important role in ovulation and might be a potential target for non‐hormonal contraception, especially in combination with PGE2 synthesis inhibitors. These findings may fill the gap in understanding the role of ABCC4 in PGE2 signaling, enhance the understanding of ovulatory disorders, and facilitate the treatment and control of fertility.

The cumulus expansion process is one of the LH mediated ovulatory processes. Hyaluronan synthase 2 (HAS2) regulates the synthesis of hyaluronic acid, the main component of the cumulus expansion process. Recently, the lncRNA HAS2 antisense RNA 1 (HAS2-AS1) was identified in our global transcriptome RNA-sequencing of novel ovulation associated genes. The role of HAS2-AS1 in HAS2 regulation w.as studied previously with contradictive results in different models but not in the ovary. Taken together the induction of HAS2-AS1 and the important role of HAS2 in the cumulus expansion process, we hypothesize that HAS2-AS1 regulate HAS2 expression and function in the ovary. Therefore we undertook to study the expression, regulation, and possible functional role of HAS2-AS1 in the human ovary. HAS2-AS1, located within the HAS2 gene that was highly regulated in our library. We found that HAS2-AS1 express mainly in cumulus cells (CCs). Furthermore, HAS2-AS1 showed low expression in immature CCs and a significant increase expression in mature CCs. Functional studies reveal that inhibition of HAS2-AS1 by siRNA caused decrease expression of HAS2. Furthermore, inhibition of HAS2-AS1 by siRNA results in decrease migration of granulosa cells. Our results suggest that HAS2-AS1 is an LH/hCG target gene that plays a positive role in HAS2 expression and thus might play a role in regulating cumulus expansion and migration.

Serum AMH is declining with age and is highly associated with ovarian follicular reserve and disordered folliculogenesis. However, the precise role of AMH in the process of human follicular aging has still to be determined. This study investigates AMH level in the follicular fluid (FF) and mRNA expression pattern in cumulus and mural granulosa cells of human ovarian follicles in relation to age. We conducted a prospective study. Sixty-eight women undergoing In vitro fertilization (IVF) treatment were enrolled in the study. We obtained FF, mural and cumulus granulosa cells from large preovulatory follicles (17-20 mm) of 21–35 years old women (n = 40) and 40–45 years old women (n = 28) during oocyte pickup. Higher level of AMH mRNA expression in cumulus cells was observed in the older age group compared to the younger (P <0.01). In accordance with AMH mRNA expression results, FF AMH protein levels were significantly higher in the older group than in the younger group (4.7 ± 1.1 ng\ml and 2.3 ± 0.2 ng\ml respectively, p < 0.002). AMH is highly expressed and secreted from cumulus GCs of advanced age patients. This remarkable correlation between AMH mRNA levels in cumulus cells in respect to age suggests that AMH may be involved in follicular aging process.