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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.

Infertility affects one in six couples, half of which are caused by a male factor. Male infertility can be caused by both, qualitative and quantitative defects, leading to Oligo- astheno-terato-zoospermia (OAT; impairment in ejaculate sperm cell concentration, motility and morphology). Azoospermia defined as complete absence of sperm cells in the ejaculation. While hundreds of genes are involved in spermatogenesis the genetic etiology of men's infertility remains incomplete.We identified a hemizygous stop gain pathogenic variation (PV) in the X-linked Germ Cell Nuclear Acidic Peptidase (GCNA), in an Azoospermic patient by exome sequencing. Assessment of the prevalence of pathogenic variations in this gene in infertile males by exome sequence data of 11 additional unrelated patients identified a probable hemizygous causative missense PV in GCNA in a severe OAT patient. Expression of GCNA in the patients' testes biopsies and the stage of spermatogonial developmental arrest were determined by immunofluorescence and immunohistochemistry. The Azoospermic patient presented spermatogenic maturation arrest with an almost complete absence of early and late primary spermatocytes and thus the complete absence of sperm. GCNA is critical for genome integrity and its loss results in genomic instability and infertility in Drosophila, C. elegans, zebrafish, and mouse. PVs in GCNA appear to be incompatible with male fertility in humans as well: A stop-gain PV caused Azoospermia and a missense PV caused severe OAT with very low fertilization rates and no pregnancy in numerous IVF treatments.

Objective To study the association among fertility treatments, treatment protocol, and offspring neoplasm risk up to the age of 18 years. Design A population-based retrospective cohort. Setting Soroka University Medical Center (SUMC), the single tertiary medical center and in vitro fertilization (IVF) unit in southern Israel. Patient(s) All offspring born at the SUMC between the years 1995 and 2018 after IVF treatment (the exposed group) and offspring conceived spontaneously (the unexposed group). Intervention(s) The study was performed at the SUMC, the single tertiary medical center and IVF unit in southern Israel. The exposed and unexposed were matched with a ratio of 1:4, based on maternal age and calendar month of delivery. Data collection included a summary of the couple’s medical records, delivery data, and offspring neoplasm diagnoses. Main Outcome Measure(s) Offspring neoplasm of any kind and time to diagnosis in each of the groups. Result(s) A total of 1,583 exposed and 5,874 offspring were included in the study. The incidences of offspring benign neoplasm were 14 (0.9%) versus 21 (0.4%), and the incidences of malignancies were 17 (1.1%) versus 29 (0.5%) among offspring of the IVF and spontaneous groups, respectively. The association between mode of conception and offspring neoplasm risk remained significant after adjusting for confounders, including mode of delivery and pregnancy complications such as hypertensive disorder, gestational diabetes mellitus, and preterm delivery compared with spontaneously conceived offspring. Among the IVF group, the increased risk for neoplasm was found among offspring who were transferred as fresh embryos, at an earlier stage of development (cleavage stage), or after three or more aspirated oocytes. Conclusion(s) IVF treatment is associated with offspring neoplasm risk; specifically, the risk was greater among offspring who were returned as fresh embryos, at an earlier embryotic stage (cleavage stage), or after three or more aspirated oocytes.

The need for pharmaceutical compounds that improve female fertility is crucial since many women that postpone childbearing today face a decline in their fertility. The essential role of telomerase in female fertility was previously demonstrated and the 4th generation of telomerase knockout mice are infertile. Therefore, increasing telomerase in the ovary may improve fertility by influencing folliculogenesis, oocyte maturation and ovulation processes. We used novel compounds (AGS) that transiently increased telomerase reverse transcriptase (TERT) expression and activity in vitro and in-vivo, to examine their effect on the folliculogenesis and ovulation processes in WT (TERT+/+) and TERT knock out (KO) mice. A single injection of AGS increased telomerase expression and activity in the WT mouse ovary, accelerated oocyte maturation and ovulation and increased the expression of fertility related genes. An increase in the plasma progesterone level and the gonadotropins hormones expression was also observed. The outcome of these enhancements is an increase in the number of embryos and offspring which exhibited a normal development and growth. In TERT KO mice AGS treatment did not affect the mouse ovary stage and the expression of specific genes. These data demonstrate the importance of the transient increase of telomerase in the ovary by pharmaceutical compounds for the improvement of female fertility.

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.

BackgroundMutationin S-phase cyclin A-associated protein rin the endoplasmic reticulum(SCAPER) have been found across ethnicities and have been shown to cause variable penetrance of an array of pathological traits, including intellectual disability, retinitis pigmentosa and ciliopathies.MethodsHuman clinical phenotyping, surgical testicular sperm extraction and testicular tissue staining. Generation and analysis ofshort spindle 3(ssp3) (SCAPERorthologue)DrosophilaCAS9-knockout lines. In vitro microtubule (MT) binding assayed by total internal reflection fluorescence microscopy.ResultsWe show that patients homozygous for aSCAPERmutation lack SCAPER expression in spermatogonia (SPG) and are azoospermic due to early defects in spermatogenesis, leading to the complete absence of meiotic cells.Interestingly,Drosophila null mutants for the ubiquitously expressedssp3gene are viable and female fertile but male sterile. We further show that male sterility inssp3null mutants is due to failure in both chromosome segregation and cytokinesis. In cells undergoing male meiosis, the MTs emanating from the centrosomes do not appear to interact properly with the chromosomes, which remain dispersed within dividing spermatocytes (SPCs). In addition, mutant SPCs are unable to assemble a normal central spindle and undergo cytokinesis. Consistent with these results, an in vitro assay demonstrated that both SCAPER and Ssp3 directly bind MTs.ConclusionsOur results show thatSCAPERnull mutations block the entry into meiosis of SPG, causing azoospermia. Null mutations inssp3specifically disrupt MT dynamics during male meiosis, leading to sterility. Moreover, both SCAPER and Ssp3 bind MTs in vitro. These results raise the intriguing possibility of a common feature between human andDrosophilameiosis.

BackgroundOligoteratoasthenozoospermia (OTA) combines deteriorated quantity, morphology and motility of the sperm, resulting in male factor infertility.MethodsWe used whole genome genotyping and exome sequencing to identify the mutation causing OTA in four men in a consanguineous Bedouin family. We expressed the normal and mutated proteins tagged with c-Myc at the carboxy termini by transfection with pCDNA3.1 plasmid constructs to evaluate the effects on protein stability in HEK293 cells and on the kinetics of actin repolymerisation in retinal pigment epithelium cells. Patients’ sperm samples were visualised by transmission electron microscopy to determine axoneme structures and were stained with fluorescent phalloidin to visualise the fibrillar (F)-actin.ResultsA homozygous missense mutation in Ciliogenesis Associated TTC17 Interacting Protein (CATIP): c. T103A, p. Phe35Ile, a gene encoding a protein important in actin organisation and ciliogenesis, was identified as the causative mutation with a LOD score of 3.25. The mutation reduces the protein stability compared with the normal protein. Furthermore, overexpression of the normal protein, but not the mutated protein, inhibits repolymerisation of actin after disruption with cytochalasin D. A high percentage of spermatozoa axonemes from patients have abnormalities, as well as disturbances in the distribution of F-actin.ConclusionThis is the first report of a recessive mutation in CATIP in humans. The identified mutation may contribute to asthenozoospermia by its involvement in actin polymerisation and on the actin cytoskeleton. A mouse knockout homozygote for CATIP was reported to demonstrate male infertility as the sole phenotype.

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 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.

Automated live embryo imaging has transformed in-vitro fertilization (IVF) into a data-intensive field. Unlike clinicians who rank embryos from the same IVF cycle cohort based on the embryos visual quality and determine how many embryos to transfer based on clinical factors, machine learning solutions usually combine these steps by optimizing for implantation prediction and using the same model for ranking the embryos within a cohort. Here we establish that this strategy can lead to sub-optimal selection of embryos. We reveal that despite enhancing implantation prediction, inclusion of clinical properties hampers ranking. Moreover, we find that ambiguous labels of failed implantations, due to either low quality embryos or poor clinical factors, confound both the optimal ranking and even implantation prediction. To overcome these limitations, we propose conceptual and practical steps to enhance machine-learning driven IVF solutions. These consist of separating the optimizing of implantation from ranking by focusing on visual properties for ranking, and reducing label ambiguity.Lay SummaryBackgroundIn vitro fertilization (IVF) is the process where a cohort of embryos are developed in a laboratory followed by selecting a few to transfer in the patient’s uterus. After approximately forty years of low-throughput, automated live embryo imaging has transformed IVF into a data-intensive field leading to the development of unbiased and automated methods that rely on machine learning for embryo assessment. These advances are now revolutionizing the field with recent retrospective papers demonstrating computational models comparable and even exceeding clinicians’ performance, startups and medical companies are securing significant funds and at advanced stages of regulatory approvals. Traditionally, embryo selection is performed by clinicians ranking cohort embryos based solely on their visual qualities to estimate implantation potential, and then using non-visual clinical properties that are common to all cohort embryos to decide how many embryos to transfer. Machine learning solutions usually combine these two steps by optimizing for implantation prediction and using the same model for ranking the embryos within a cohort under the implicit assumption that training to predict implantation potential also optimizes a solution to the problem of ranking embryos from a specific cohort.ResultsIn this multi-center retrospective study we analyzed over 48,000 live imaged embryos to provide evidence that the common machine-learning scheme of training a model to predict implantation and using the same model for embryo ranking is wrong. We made this point by explicitly decoupling the problems of embryo implantation prediction and ranking with a set of computational analyses. We demonstrated that: (1) Using clinical cohort-related information (oocyte age) improves embryo implantation prediction but deteriorates ranking, and that (2) The label ambiguity of the embryos that failed to implant (it is not known whether the embryo or the external factors were the reason for failure) deteriorates embryo ranking and even the ability to accurately predict implantation. Our study provides a quantitative mapping of the tradeoffs between data volume, label ambiguity and embryo quality. In a key result, we reveal that considering embryos that were excluded based on their poor visual appearance (called discarded embryos), although commonly thought as trivially discriminated from high quality embryos, enhances embryo ranking by reducing the ambiguity in their (negative) labels. These results establish the benefit of harnessing the availability of extensive data and reliable labels in discarded embryos to improve embryo ranking and implantation prediction.OutlookWe make two practical recommendations for devising machine learning solutions to embryo selection that will open the door for future advancements by data scientists and IVF technology developers. Namely, training models for embryo ranking should: (1) focus exclusively on embryo intrinsic features. (2) include less ambiguous negative labels, such as discarded embryos. In the era of machine learning, these guidelines will shift back the traditional two-step process of optimizing embryo ranking and implantation prediction independently under the appropriate assumptions - an approach better reflecting the clinician’s decision that involves the evaluation of all the embryos in the context of its cohort.

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

Study question Does high ovarian response to controlled ovarian stimulation (COS) have a negative effect on oocyte quality? Summary answer High ovarian response is associated with reduced oocyte quality manifested as higher fraction of immature oocytes and higher rate of direct uneven cleavage (DUC) embryos. What is known already The literature regarding the effect of ovarian hyperstimulation on oocyte quality is limited and controversial. Results from several studies suggest that hyper response to controlled ovarian stimulation has a detrimental effect on oocyte and embryo quality, while others failed to confirm the existence of a direct toxic effect on oocyte and embryo quality. The association between temporal embryonal milestones and implantation rate has been previously demonstrated ,offering an additive tool by which oocyte quality, represented by embryo dynamics, can be evaluated. None of the aforementioned studies, however, used time lapse monitoring (TLM) system to evaluate oocyte and embryo quality. Study design, size, duration This study included a retrospective assessment of morphokinetic parameters performed by TLM from three university affiliated medical centers between January 2014 and December 2019. The developmental process and kinetics of 1863 embryos obtained from the study group, referred as the “ high ovarian response” (HOR) group, was compared to 4907 embryos from the control group - the “normal ovarian response” (NOR) group. Participants/materials, setting, methods The study included patients younger than 38 years who underwent COS with consecutive aspiration of either more than 15 oocytes (the HOR group) or 6–15 oocytes (the NOR group). A comparison was made between the groups regarding morphokinetic parameters, including the rate of embryos manifesting direct uneven cleavage (DUC) at first cleavage (DUC–1), as well as implantation and pregnancy rates. Logistic regression was conducted to assess the association between patients’ characteristics and implantation rate. Main results and the role of chance Oocyte maturation rate was significantly lower, and the DUC–1 embryos rate was significantly higher in the high ovarian response group compared to the normal ovarian response group (56.5% Vs 90.0%, p Limitations, reasons for caution The groups were not homogenous in their basic characteristics. Important information regarding the maximal dose of GT obtained, previous IVF response and ovarian reserve testing was lacking Wider implications of the findings: Higher oocyte quantity might have an effect on oocyte quality manifested as higher fraction of incompetent oocytes and higher rate of DUC–1 embryos. Once beyond the preliminary developmental stages, embryos from both groups reach the morphokinetic milestones at a similar rate and display similar implantation and pregnancy rates. Trial registration number Not applicable

PURPOSE: To assess oocyte quality in young patients with decreased ovarian response to controlled ovarian stimulation using time-lapse analysis. METHODS: A retrospective cohort study conducted at five medical centers between 2013 and 2017. The “decreased ovarian response” (DOR) group consisted of 241 women who underwent controlled ovarian stimulation with ≤ 5 retrieved oocytes and 519 cultured embryos. The “normal response” (NOR) group consisted of 667 women with ≥ 6 retrieved oocytes resulting in 3633 embryos. Data included annotation of morphokinetic events of embryos cultured in a time-lapse incubator from time of pronuclei appearance to time of starting blastocyst formation (tSB). Comparison was made between morphokinetic parameters of DOR and NOR patients with additional subgroup analysis according to the implantation status. RESULTS: Implantation and clinical pregnancy rates were significantly higher in the NOR group compared with the DOR group (44.5% vs. 31.6% and 51.5% vs. 37.7%, respectively; p < 0.05). Embryos from the DOR group reached the morphokinetic milestones later than embryos obtained from NOR patients. In the DOR group, implanted embryos reached starting blastocyst formation (tSB) faster than embryos which failed to be implanted, however, manifested a protracted course compared with implanted embryos from the NOR group. In a multivariate analysis—decreased ovarian response, nulliparity, number of transferred embryos, and t4, and were predictive for implantation. CONCLUSIONS: The quantitative decrease in ovarian response is associated with reduced oocyte quality, reflected by a slower developmental rate and lower implantation and pregnancy rates. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s10815-021-02113-4.

Background: The need for a reliable prediction model of testicular sperm extraction outcomes in non-obstructive azoospermia patients is inarguably of paramount importance. The aim of the present study was to determine whether a combination of available parameters would improve the prediction of successful sperm retrieval. Methods: A retrospective cohort study with a population of 119 patients who underwent Testicular Sperm Extraction - was conducted. We used univariate and multivariate binary logistic regression to identify the most reliable factors associated with the success of the surgical procedure. Results: Age and ethnicity were the only parameters that differed significantly between the patients who experienced successful testicular sperm extraction and those who did not. A comparison between patients of Jewish and Bedouin origin demonstrated that despite the fact that the Jewish population was significantly older, the sperm retrieval rate was much higher than it was in the Bedouin population-77.19% versus 54.88%. Conclusion: To the best of our knowledge, this is the first study to emphasize ethnic origin as an important parameter in the prediction of successful sperm retrieval in non-obstructive azoospermia patients.

STUDY QUESTION Can a machine-learning-based model trained in clinical and biological variables support the prediction of the presence or absence of sperm in testicular biopsy in non-obstructive azoospermia (NOA) patients? SUMMARY ANSWER Our machine-learning model was able to accurately predict (AUC of 0.8) the presence or absence of spermatozoa in patients with NOA. WHAT IS KNOWN ALREADY Patients with NOA can conceive with their own biological gametes using ICSI in combination with successful testicular sperm extraction (TESE). Testicular sperm retrieval is successful in up to 50% of men with NOA. However, to the best of our knowledge, there is no existing model that can accurately predict the success of sperm retrieval in TESE. Moreover, machine-learning has never been used for this purpose. STUDY DESIGN, SIZE, DURATION A retrospective cohort study of 119 patients who underwent TESE in a single IVF unit between 1995 and 2017 was conducted. All patients with NOA who underwent TESE during their fertility treatments were included. The development of gradient-boosted trees (GBTs) aimed to predict the presence or absence of spermatozoa in patients with NOA. The accuracy of these GBTs was then compared to a similar multivariate logistic regression model (MvLRM). PARTICIPANTS/MATERIALS, SETTING, METHODS We employed univariate and multivariate binary logistic regression models to predict the probability of successful TESE using a dataset from a retrospective cohort. In addition, we examined various ensemble machine-learning models (GBT and random forest) and evaluated their predictive performance using the leave-one-out cross-validation procedure. A cutoff value for successful/unsuccessful TESE was calculated with receiver operating characteristic (ROC) curve analysis. MAIN RESULTS AND THE ROLE OF CHANCE ROC analysis resulted in an AUC of 0.807 ± 0.032 (95% CI 0.743-0.871) for the proposed GBTs and 0.75 ± 0.052 (95% CI 0.65-0.85) for the MvLRM for the prediction of presence or absence of spermatozoa in patients with NOA. The GBT approach and the MvLRM yielded a sensitivity of 91% vs. 97%, respectively, but the GBT approach has a specificity of 51% compared with 25% for the MvLRM. A total of 78 (65.3%) men with NOA experienced successful TESE. FSH, LH, testosterone, semen volume, age, BMI, ethnicity and testicular size on clinical evaluation were included in these models. LIMITATIONS, REASONS FOR CAUTION This study is a retrospective cohort study, with all the associated inherent biases of such studies. This model was used only for TESE, since micro-TESE is not performed at our center. WIDER IMPLICATIONS OF THE FINDINGS Machine-learning models may lay the foundation for a decision support system for clinicians together with their NOA patients concerning TESE. The findings of this study should be confirmed with further larger and prospective studies. STUDY FUNDING/COMPETING INTEREST(S) The study was funded by the Division of Obstetrics and Gynecology, Soroka University Medical Center, there are no potential conflicts of interest for all authors.

The present study investigated the association between oocyte zona pellucida shear modulus (ZPSM) and implantation rate (IR). Ninety-three oocytes collected from 38 in-vitro fertilization patients who underwent intracytoplasmic sperm injection were included in this case–control study. The ZP was modeled as an isotropic compressible hyperelastic material with parameter $$C_{10}$$ C 10 , which represents the ZPSM. Computational methodology was used to calculate the mechanical parameters that govern ZP deformation. Fifty-one developed embryos were transferred and divided into two groups—implanted and not implanted. Multivariate logistic regression analysis was performed to identify the association between ZPSM and IR while controlling for confounders. Maternal age and number of embryos per transfer were significantly associated with implantation. The IR of embryos characterized by $$C_{10}$$ C 10 values in the range of 0.20–0.40 kPa was 66.75%, while outside this range it was 6.70%. This range was significantly associated with implantation (p p = 0.001). The present study suggests that ZPSM may improve the classic embryo selection process with the aim of increasing IR.

Background Ubiquitin‐Specific Peptidase 26 (USP26), located on the X chromosome, encodes a deubiquitinating enzyme expressed mainly in testis, where it regulates protein turnover during spermatogenesis and modulates the ubiquitination levels of the Androgen Receptor (AR), and as a consequence, affects AR signaling. Methods The patient was thoroughly characterized clinically. He was genetically tested by chromosome analysis and whole exome sequencing (WES). Results The patient was diagnosed with Sertoli cell‐only syndrome pattern (SCOS). The WES analysis revealed only the variation in USP26: causing p.P469S in a highly evolutionary conserved amino acid as the possible cause for SCOS. The literature search identified 34 single variations and 14 clusters of variations in USP26 that were associated with male infertility. Only one of the 22 variations and of one cluster of three mutations tested for ubiquitination activity was found as damaging. Only one out of six variations tested for effect on AR function was found as damaging. Thus, the association of USP26 with male fertility was questioned. Conclusions The finding in our patient and the discussion on the reviewed literature support a possible role for USP26 in male fertility., Many mutations in USP26 were reported to be causing azoospermia. However, the role of inactivation of this gene as causing azoospermia has been controversial. We present a novel mutation in a Sertoli cell‐only syndrome patient and critically review the previous reports, supporting the role of functional USP26 for male fertility.

MutationHuman clinical phenotyping, surgical testicular sperm extraction and testicular tissue staining. Generation and analysis ofWe show that patients homozygous for aOur results show that

Oligoteratoasthenozoospermia (OTA) combines deteriorated quantity, morphology and motility of the sperm, resulting in male factor infertility.We used whole genome genotyping and exome sequencing to identify the mutation causing OTA in four men in a consanguineous Bedouin family. We expressed the normal and mutated proteins tagged with c-Myc at the carboxy termini by transfection with pCDNA3.1 plasmid constructs to evaluate the effects on protein stability in HEK293 cells and on the kinetics of actin repolymerisation in retinal pigment epithelium cells. Patients' sperm samples were visualised by transmission electron microscopy to determine axoneme structures and were stained with fluorescent phalloidin to visualise the fibrillar (F)-actin.A homozygous missense mutation in Ciliogenesis Associated TTC17 Interacting Protein (This is the first report of a recessive mutation in

In in vitro fertilization (IVF) treatments, early identification of embryos with high implantation potential is required for shortening time to pregnancy while avoiding clinical complications to the newborn and the mother caused by multiple pregnancies. Current classification tools are based on morphological and morphokinetic parameters that are manually annotated using time‐lapse video files. However, manual annotation introduces interobserver and intraobserver variability and provides a discrete representation of preimplantation development while ignoring dynamic features that are associated with embryo quality. A fully automated and standardized classifiers are developed by training deep neural networks directly on the raw video files of >6200 blastulation‐labeled and >5500 implantation‐labeled embryos. Prediction of embryo implantation is more accurate than the current state‐of‐the‐art morphokientic classifier. Embryo classification improves with video length where the most predictive images show only partial association with morphological features. Deep learning substitute to human evaluation of embryo developmental competence thus contributes to implementing single embryo transfer methodology.

The aim of this study was to compare the in vitro fertilization (IVF) cycles ended by miscarriage with subsequent IVF cycles in relation to various IVF cycle parameters and pregnancy termination modalities. Comparison of pre-miscarriage to post-miscarriage IVF cycles parameters demonstrated that lower peak E2 levels (1087 ± 593 versus 1237 ± 676 pg/ml, respectively; p < 0.05) were achieved in the post miscarriage cycles despite higher total gonadotropin consumption (2341 ± 1488 versus 2115 ± 1101 IU; p

Background Azoospermia is diagnosed when sperm cells are completely absent in the ejaculate even after centrifugation. It is identified in approximately 1% of all men and in 10%–20% of infertile males. Non-obstructive azoospermia (NOA) is characterised by the absence of sperm due to either a Sertoli cell-only pattern, maturation arrest, hypospermatogenesis or mixed patterns. NOA is a severe form of male infertility, with limited treatment options and low fertility success rates. In the majority of patients, the cause for NOA is not known and mutations in only a few genes were shown to be causative. Aim We investigated the cause of maturation arrest in five azoospermic infertile men of a large consanguineous Bedouin family. Methods and results Using whole genome genotyping and exome sequencing we identified a 4 bp deletion frameshift mutation in TDRD9 as the causative mutation with a Lod Score of 3.42. We demonstrate that the mutation results in a frameshift as well as exon skipping. Immunofluorescent staining with anti-TDRD9 antibody directed towards the N terminus demonstrated the presence of the protein in testicular biopsies of patients with an intracellular distribution comparable to a control biopsy. The mutation does not cause female infertility. Conclusion This is the first report of a recessive deleterious mutation in TDRD9 in humans. The clinical phenotype recapitulates that observed in the Tdrd9 knockout mice where this gene was demonstrated to participate in long interspersed element-1 retrotransposon silencing. If this function is preserved in human, our data underscore the importance of maintaining DNA stability in the human male germ line.

Poor ovarian response (POR)—retrieval of 3 or fewer eggs, is a challenging issue in IVF. A retrospective study included POR women who underwent 386 IVF cycles. The data were classified in four groups according to women’s age (years) during the treatment cycle: 1) 20 - 34 (n = 133), 2) 35 - 39 (n = 133), 3) 40 - 42 (n = 78), 4) 43 - 47 y (n = 42), and correlated with the characteristics of the population. The clinical pregnancy rates for groups 1, 2, 3, and 4 were: 23.3%, 12%, 2.6%, 4.8%, respectively. It was found to be significantly higher (p

Objective To compare intracytoplasmic sperm injection (ICSI) outcomes with the use of fresh or frozen-thawed ejaculated or testicular sperm in patients with cryptozoospermia or nonobstructive azoospermia. Design Retrospective cohort study. Setting Tertiary medical center IVF unit. Patient(s) A total of 274 patients evaluated from 1999 to 2011. Intervention(s) A total of 103 patients underwent testicular sperm extraction (TESE) because of nonobstructive azoospermia, and 171 patients were diagnosed with cryptozoospermia. Main Outcome Measure(s) ICSI outcomes during the first cycle in each technique performed according to the sperm origin (testicular vs. ejaculated) and processing (frozen vs. fresh). Result(s) Forty-eight cycles with the use of frozen testicular sperm, 22 cycles with fresh testicular sperm, 66 cycles with frozen ejaculated sperm, and 138 cycles with fresh ejaculated sperm were examined. Significantly more motile sperm were found in the fresh ejaculate group compared with the frozen-thawed ejaculate group (96% vs. 88%, respectively). Furthermore, fresh ejaculated sperm were found to have better fertilization rates than frozen ejaculated sperm (64% vs. 56%, respectively). No significant difference was found between fresh and frozen-thawed testicular sperm, either in motile sperm available for ICSI or in fertilization rate (64% vs. 62% and 52% vs. 49%, respectively). Conclusion(s) In cases of cryptozoospermia, frozen-thawed ejaculated sperm is inferior to fresh ejaculated sperm in fertilization rates. However, in nonobstructive azoospermia, no major differences were found between fresh and frozen-thawed testicular sperm. Therefore, uncoupled TESE/oocyte pick-up (OPU) should be considered in NOA cases to prevent possible unnecessary ovarian stimulation and OPU when no sperm cells are detected.

Oxidative stress is induced by reactive oxygen substances (ROS) that are known to affect male fertility. The aims of this study were to prospectively investigate and characterize total antioxidant and specifically polyphenols concentrations and their relations to sperm quality and fertility treatment success. During their infertility treatment, sixty-seven males were prospectively recruited to this study. After separation of the sperm from the semen sample, the semen fluid samples antioxidants and polyphenols concentrations were determined. Antioxidant concentration was significantly associated with sperm concentration and total motile count. Antioxidants concentration in the group of male with sperm concentration ≥ 15 × 106was significantly higher than in the group of male with antioxidants concentration < 15 × 106(830.3 ± 350 μM and 268.3 ± 220 μM, resp.,p<0.001). Polyphenols concentration did not differ between the groups of sperm concentration above and below 15 × 106(178.7 ± 121 μM and 161.7 ± 61 μM, resp.,p-NS). No difference was found between fertilization rates and antioxidants or polyphenols concentrations. This is the first study that reports on polyphenols concentration within semen fluid. More studies are needed in order to investigate polyphenols role in male fertility.

Background The condensed state of the human sperm's chromatin is essential for the compact structure of the spermatozoon head, which is important for the fertilization process. The enzymes DNA topoisomerases (topo I and topo II) are responsible for the topological structure of the chromatin in somatic cells. The activities and the characterization of topoisomerases in mature human sperm cells have not been previously investigated. Methods Sperm cells were purified from the semen of healthy donors by standard procedures and assays measuring the activities, protein size and sensitivity to inhibitors of topoisomerases were performed. Results Topo I and topo II DNA relaxation activities are present in nuclear extracts derived from human sperm. The sperm topo I activity is inhibited by camptothecin, similarly to the somatic enzyme. An 85 kDa sperm protein, compared with the 100 kDa somatic topo IB enzyme, reacted with anti-human topo I antibody. Sperm topo II lacks the DNA decatenation activity of the somatic enzyme and a 97 kDa protein, compared with the 170 kDa somatic topo IIalpha enzyme, was detected with anti-human topo II antibody. Sperm nuclear extracts contained inhibitors of somatic topo II decatenation activity. Conclusions Topoisomerase I and II activities as well as topo I and topo II proteins are present in mature human sperm cells. These enzymes possess unique properties compared with their somatic counterparts.

The current research is aimed at finding potential non-invasive bio-markers that will help us learn more about the mechanisms at play in failed assisted reproduction treatment. This exploratory pilot study examined the relationship between cell-free DNA (CFD) in plasma and telomere length in lymphocytes among women undergoing in vitro fertilization (IVF) and compared telomere length and CFD levels to a healthy control group.Blood of 20 women undergoing IVF was collected at three time points during the IVF cycle. We assessed the relationship between CFD and telomere length as well as controlling for morning cortisol levels. We also collected blood of 10 healthy controls at two time points (luteal and follicular phases of the menstrual cycle) and compared mean telomere length, CFD, and cortisol levels between the IVF patients and healthy controls.The results revealed an inverse relationship between CFD levels and telomere lengths at several time points that remained significant even after controlling for cortisol levels. Women undergoing IVF had statistically significant higher levels of CFD and shorter telomeres compared to healthy controls.The relationship between telomere length and CFD should be further explored in larger studies in order to uncover potential mechanisms that cause both shortened telomere length and elevated CFD in women undergoing IVF.

Thirty-five pregnancies were inadvertently conceived during the midluteal application of the long-acting gonadotropin-releasing hormone agonist triptorelin acetate before ovarian stimulation for in vitro fertilization. Most of the conceptions occurred before the first or second IVF trial, and were associated with a significantly increased tendency to develop gestational diabetes. No increase in pregnancy wastage or appearance of congenital malformations occurred.

Introduction. To report a live birth following egg retrieval after only 12 hours from hCG priming.Patients. A childless couple with five-years-lasting secondary infertility.Methods. IVF was performed according to the long protocol. Two immature oocytes were retrieved following only 12 hours after hCG priming due to the patient misunderstanding. The eggs were culturedin vitroand ICSI was performed following polar body extruded after 24 hours in culture. After additional 24 hours a 4-cell embryo was developed and ET was performed.Results. A viable pregnancy was achieved and a healthy baby girl was delivered at 38 weeks of gestation.Conclusion. In a rare and unexpected situation when immature oocytes are retrieved following a short hCG priming, the eggs should be culturedin vitro,late ICSI should be performed, and a pregnancy may be expected.

This prospective repeated measures study was designed to examine the cell-free DNA (cfDNA) concentrations during ovarian stimulation and the relationship between cfDNA concentration and pregnancy rates in women undergoing IVF-embryo transfer. The study examined 37 women undergoing IVF treatment in an IVF unit in a university medical centre in southern Israel. cfDNA concentrations were measured by a direct fluorescence assay, pregnancy rates were identified by plasma β human chorionic gonadotrophin (HCG) concentrations and verified by vaginal ultrasound to determine gestational sac and fetal heart beats. Throughout the IVF cycle, at the three time points measured, the mean concentration of plasma cfDNA among all participants did not statistically significantly change. However, on the day of βHCG test in patients undergoing IVF-embryo transfer, plasma cfDNA concentrations were statistically significantly higher among women who did not conceive in comparison to those who conceived. Plasma cfDNA may reflect the presence of factors which interfere with embryo implantation. Further research is required to determine the usefulness of cfDNA as a biomarker of IVF outcome and to examine the underlying pathologies as potential sources for increased plasma cfDNA concentrations. Cell-free DNA (cfDNA) is particles of DNA which are released from the cell nucleus and are found in high concentrations during a variety of illnesses and injuries. This study was designed to examine the cfDNA concentrations during IVF treatment and the relationship between cfDNA concentration in the bloodstream and pregnancy rates in women undergoing IVF. This study examined 37 women in treatment at the IVF unit of the University Medical Centre in southern Israel. cfDNA concentrations in the bloodstream were measured at three time points by a direct test. Pregnancy rates were identified by pregnancy hormone concentrations in the bloodstream and verified by vaginal ultrasound to determine a pregnancy sac and fetal heart beats. Throughout the IVF cycle, at the three time points measured, the average concentration of cfDNA among all participants did not change. However, on the day of the pregnancy test, blood cfDNA concentrations were significantly higher among women who were not pregnant in comparison to those who were. Plasma cfDNA may reflect the presence of factors which interfere with embryo implantation. Further research is required to determine the usefulness of cfDNA as a biological marker of IVF outcome and examine underlying illnesses and problems as potentials sources for increased cfDNA concentrations.

Objective To assess whether the pronuclear score and embryo nuclear scoring have an additive value to day 2 morphology criteria in the prediction of the implantation rate (IR). Design Retrospective clinical study. Setting IVF Unit, Soroka University Medical Center. Patient(s) Analysis of all homogenous IVF fresh embryo transfers from 2008–2010. Intervention(s) Morphological evaluation of pronuclear scoring, embryo nuclear scoring, and day 2 embryo morphology scoring on embryos obtained from IVF or intracytoplasmic sperm injection (ICSI) cycles. Main Outcome Measure(s) Implantation rate of homogeneous transfers. Result(s) No statistical association was found between the pronuclear scoring system (Z) and IR. The pronuclear score Z3 had a significantly better performance than Z1, Z2, and Z4 (IR = 25.2%, 22%, 21.4%, and 11.8%, respectively). Assessment of the second day embryo morphology scoring (index [IDX]) was found to associate with IR. IDX1, IDX2, IDX3, IDX4, and IDX5 had an IR of 36.1%, 28%, 21.9%, 14.2%, and 0, respectively. The nuclear scoring including: 1 nucleus in all blastomeres, 1 nucleus in part of the blastomeres, no visualization, and multinucleation showed high association with IR=32.7%, 22.9%, 14.8%, and 9.1%, respectively. The effect of the nuclear scoring on IR was lost in a multivariable analysis that included day 2 embryo morphology scoring. Conclusion(s) Our results suggest that there is no additive value in grading the zygote on day 1 or embryo nuclear scoring on day 2 to day 2 embryo morphology for the prediction of IR.

Background Embryo implantation plays a major role in embryogenesis and the outcome of pregnancy. Plasminogen activators (PAs) have been implicated in mammalian fertilization, early stages of development and embryo implantation. As in-vitro developing embryos resulted in lower implantation rate than those developed in-vivo we assume that a reduced PAs activity may be involved. In the present work we studied the effect of EGF on PAs activity, quantity and embryo implantation. Methods Zygotes were flushed from rat oviducts on day one of pregnancy and grown in-vitro in R1ECM supplemented with EGF (10 ng/ml) and were grown up to the blastocyst stage. The control groups were grown in the same medium without EGF. The distribution and quantity of the PAs were examined using fluorescence immunohistochemistry followed by measurement of PAs activity using the chromogenic assay. Implantation rate was studied using the embryo donation model. Results PAs distribution in the embryos was the same in EGF treated and untreated embryos. Both PAs were localized in the blastocysts' trophectoderm, supporting the assumption that PAs play a role in the implantation process in rats. EGF increased the quantity of uPA at all stages studied but the 8-cell stage as compared with controls. The tissue type PA (tPA) content was unaffected except the 8-cell stage, which was increased. The activity of uPA increased gradually towards the blastocyst stage and more so due to the presence of EGF. The activity of tPA did not vary with the advancing developmental stages although it was also increased by EGF. The presence of EGF during the preimplantation development doubled the rate of implantation of the treated group as compared with controls.

Objective To evaluate the relationship between duration of sexual abstinence and various characteristics of normal and subnormal semen. Design A retrospective study based on computerized data. Setting Fertility and IVF unit at a university medical center. Patient(s) Nine thousand, four hundred eighty-nine semen samples from 6,008 patients were analyzed according to the World Health Organization (WHO) manual and grouped according to sperm concentration (106/mL) into severe (0.2–4 × 106), moderate (>4–10 × 106), and mild (>10–19.99 × 106) oligozoospermia, and normozoospermia (≥20–250 × 106) groups. Main Outcome Measure(s) In each group mean values of semen volume, sperm concentration, percentage of motile sperm and of normal morphology (according to WHO or Kruger criteria), total sperm count, and total motile sperm count per ejaculate were related to duration of abstinence. Result(s) Among the 3,506 oligozoospermic samples, the peak mean sperm motility of 30.3% was observed after 1 day of abstinence. Similarly, the mean percentage of normal morphology among mild-moderate oligozoospermic samples (n = 2,260) reached peak values of 7.4%–8.6% between 0–2 days of abstinence. The 5,983 normozoospermic samples showed a significant decrease in the percentage of sperm motility and normal morphology to mean values of 33.1% and 7.0%, respectively, on days 11–14 of sexual abstinence. Conclusion(s) Our data challenge the role of abstinence in male infertility treatments and suggest that to present the best possible semen samples, patients with male factor infertility should collect the semen after just 1 day of sexual abstinence. Patients presenting normal sperm analysis or sperm donors for cryopreservation purposes should be advised not to exceed 10 days of sexual abstinence.

Objective To assess the participation of urokinase-type (uPA) and tissue-type (tPA) plasminogen activators in embryo development and implantation. Design The study was set to compare the activities of PAs in embryos developed in vivo and in vitro and their implantation rates. Endometrial PA activity was studied as well. Setting University laboratories. Patient(s) None. Intervention(s) None. Main outcome measure(s) Plasminogen activator activity in embryos. Result(s) Whereas tPA activity in in vivo-developed embryos decreased from the zygote to the blastocyst stages, it did not change in embryos developed in vitro. The activity of uPA was lowest in two-cell-stage embryos, both in vivo and in vitro, and increased as embryos developed into blastocysts. The activity of uPA in the in vitro-developed embroys was lower in all developmental stages as compared with those developed in vivo. Whereas endometrial tPA activity did not change during the preimplantation period, uPA activity increased gradually toward the time of implantation. The implantation rate of the in vitro-developed embryos was much lower than that of those developed in vivo. Conclusion(s) Taken together, these data indicate that in vitro embryo development leads to lower PA activities as compared with in vivo development. The increased activity of uPA toward the blastocyst stage supports uPA's role in the implantation process. Moreover, the rise in endometrial uPA might also indicate its importance in this process.

Objective To compare blastocyst-stage embryo transfers (ETs) with day 2–3 ETs in patients who failed to conceive in three or more day 2–3 IVF/ET cycles. Design Prospective, randomized. Setting Fertility unit in a university medical center. Patient(s) Fifty-four patients with an adequate ovarian response underwent oocyte retrievals. The patients were prospectively and randomly divided into blastocyst-stage and day 2–3 ET groups. Intervention(s) Ovarian down-regulation was obtained using GnRH agonist, and controlled ovarian hyperstimulation was achieved using daily administration of gonadotropins. Main outcome measure(s) The rate of blastocyst formation, ET cancellations, pregnancies, implantation, multiple gestation, and live births. Result(s) The clinical pregnancy rates per oocyte retrieval were 21.7% and 12.9% per blastocyst and day 2–3 ETs, respectively. Although there was a significantly higher implantation rate for blastocyst embryos (21.2%) as compared with 48- to 72-hour embryos (6%), the multiple-pregnancy rate was not significantly different between both groups. An ET cancellation rate of 26% and 6.4% for blastocyst and day 2–3 ETs, respectively, was observed. The presence of two or more 8-cell embryos on day 3 in culture carried a high probability of obtaining blastocysts for transfer. Conclusion(s) This prospective randomized study suggests that in patients with an adequate ovarian response who failed to conceive in at least three IVF/ET cycles [1] transfer of blastocyst-stage embryos carries a significantly higher implantation rate; [2] the pregnancy rate per oocyte retrieval and ET are higher in the blastocyst-stage group, even if it did not reach statistical significance; [3] a higher ET cancellation rate was observed in the whole blastocyst-stage group; [4] the ET cancellation rate was reduced significantly if the decision to proceed to blastocyst transfer was made on day 3 after oocyte retrieval, which is a post hoc conclusion.