Evaluation of an artificial intelligence-facilitated sperm detection tool in azoospermic samples for use in ICSI.

RESEARCH QUESTION: Can artificial intelligence (AI) improve the efficiency and efficacy of sperm searches in azoospermic samples? DESIGN: This two-phase proof-of-concept study began with a training phase using eight azoospermic patients (>10,000 sperm images) to provide a variety of surgically collected samples for sperm morphology and debris variation to train a convolutional neural network to identify spermatozoa. Second, side-by-side testing was undertaken on two cohorts of non-obstructive azoospermia patient samples: an embryologist versus the AI identifying all the spermatozoa in the still images (cohort 1, n = 4), and a side-by-side test with a simulated clinical deployment of the AI model with an intracytoplasmic sperm injection microscope and the embryologist performing a search with and without the aid of the AI (cohort 2, n = 4). RESULTS: In cohort 1, the AI model showed an improvement in the time taken to identify all the spermatozoa per field of view (0.02 ± 0.30  ×  10-5s versus 36.10 ± 1.18s, P < 0.0001) and improved recall (91.95 ± 0.81% versus 86.52 ± 1.34%, P < 0.001) compared with an embryologist. From a total of 2660 spermatozoa to find in all the samples combined, 1937 were found by an embryologist and 1997 were found by the AI in less than 1000th of the time. In cohort 2, the AI-aided embryologist took significantly less time per droplet (98.90 ± 3.19 s versus 168.7 ± 7.84 s, P < 0.0001) and found 1396 spermatozoa, while 1274 were found without AI, although no significant difference was observed. CONCLUSIONS: AI-powered image analysis has the potential for seamless integration into laboratory workflows, to reduce the time to identify and isolate spermatozoa from surgical sperm samples from hours to minutes, thus increasing success rates from these treatments.

Temperature fluctuations during embryo transfer can be mitigated by optimizing transfer protocol.

RESEARCH QUESTION: What impact do variations in embryo transfer catheter loading and movement procedures have on temperature and pH fluctuations during embryo transfer? DESIGN: Mock embryo transfers were conducted to test the impact of air flow/movement, use of catheter coverings, and the type of workstation used for catheter loading on catheter temperature. A thermocouple probe inserted into the tip of the outer catheter or taped to the exterior of the inner catheter recorded temperature within the catheter every 5 s from time of mock embryo loading (TL) to 60 s (TL + 60 s) or from the start of transit (TT). Fluctuations in culture medium pH in embryo transfer dishes were monitored. RESULTS: The rate of cooling during transit was faster (all P < 0.05) when catheters were uncovered compared with all covering methods tested. This resulted in a lower catheter temperature at TL + 20 s (28.43 ± 0.30 °C) compared with catheters covered by plastic tubing (31.4 ± 0.30 °C), paper (31.0 ± 0.26 °C) or paper + thumb (31.1 ± 0.78 °C; all P ≤ 0.05). Temperature was maintained more effectively when catheters were loaded in a crib compared with a heated stage, until initiation of transit, when the rate of temperature decrease was similar. Culture medium pH increased more rapidly when embryo transfer dishes remained on a heated stage during the procedure compared with in an open crib. CONCLUSIONS: Temperature loss during the embryo transfer procedure can be mitigated by reducing the transit time and using catheter coverings. Use of a crib for catheter loading only improved temperature stability while the catheter remained in the crib, not during transit, and reduced pH fluctuations during the procedure.

A maternal ketogenic diet alters oviduct fluid nutrients and embryo histone acetylation in mice.

In brief: A ketogenic diet (KD) elevates blood ß-hydroxybutyrate to concentrations that are known to perturb the development, metabolism, histone acetylation and viability of preimplantation mouse embryos in culture. This study shows that a maternal KD changes available nutrient levels in the oviduct, leading to altered embryo development and epigenetic state in vivo. Abstract: A ketogenic diet elevates blood ß-hydroxybutyrate to concentrations that perturb the development, metabolism, histone acetylation (H3K27ac) and viability of preimplantation mouse embryos in vitro. However, whether a ketogenic diet alters ß-hydroxybutyrate concentrations within female reproductive fluid is unknown. This study aimed to quantify glucose and ß-hydroxybutyrate within mouse blood and oviduct fluid following standard diet and ketogenic diet consumption and to assess whether a maternal periconceptional ketogenic diet impacts in vivo embryo development and blastocyst H3K27ac. Female C57BL/6 × CBA mice were fed a standard or ketogenic diet (n = 24 each) for 24-27 days. Glucose and ß-hydroxybutyrate were quantified in blood via an electronic monitoring system and in oviduct fluid via ultramicrofluorescence. The developmental grade of flushed blastocysts was recorded, and blastocyst cell number and H3K27ac were assessed via immunofluorescence. A maternal ketogenic diet elevated ß-hydroxybutyrate in day 24 blood (P < 0.001) and oviduct fluid (P < 0.05) compared with a standard diet, whereas glucose was unchanged. A periconceptional ketogenic diet did not impact blastocyst cell number; however, it significantly delayed blastocyst development (P < 0.05) and reduced trophectoderm-specific H3K27ac (P < 0.05) compared with standard diet-derived embryos. Maternal ketogenic diet consumption is, therefore, associated with reproductive tract nutrient changes and altered embryonic development and epigenetics in vivo. Future studies to assess whether periconceptional/gestational ketogenic diet consumption impacts human preimplantation, fetal, and long-term offspring development and health are warranted.

A step closer to parenthood with non-obstructive azoospermia: Unveiling the impact of microdissection testicular sperm extraction in Australia's largest single-centre study.

BACKGROUND: Non-obstructive azoospermia (NOA) diagnosis poses challenges for couples seeking parenthood. Microdissection testicular sperm extraction (MD-TESE) excels in retrieving testicular sperm cells for NOA cases. However, limited live birth data in Australian NOA patients hinders accurate counselling. AIMS: This study aimed to determine the likelihood of infertile couples with a male partner diagnosed with NOA conceiving biological children using MD-TESE / intracytoplasmic sperm injection (ICSI). MATERIALS AND METHODS: A retrospective cohort study included 108 NOA men treated at a public fertility unit and a private fertility centre (May 2009-May 2022). PRIMARY OUTCOME: live birth rate (LBR); secondary outcomes: sperm retrieval rate, pregnancy rate, and neonatal outcomes. RESULTS: Among 108 patients undergoing MD-TESE, the positive sperm retrieval rate (PSRR) was 64.8% (70/108). Histology best predicted sperm retrieval success, with hypo-spermatogenesis yielding a 94.1% PSRR. Age, testicular volume, and hormonal parameters had no significant impact. Mean male age: 35.4 years; mean partner age: 32.7 years. Fertilisation rate: 50.7%. LBR per initiated cycle: 58.7% (37/63); per embryo transfer: 63.8% (37/58); per initially diagnosed NOA man: 34.3% (37/108). Cumulative LBR: 74.1% (43/58); twin rate: 10.8% (4/37). No neonatal deaths or defects were observed among 47 live offspring. CONCLUSION: This study provides valuable data for counselling NOA couples on the probability of conceiving biological offspring. MD-TESE and ICSI yielded favourable PSRR (64.8%) and LBR (63.8%). However, couples should be aware that once NOA is confirmed, the chance of taking home a baby is 34%.

A micro-fabricated device (microICSI) improves porcine blastocyst development and procedural efficiency for both porcine intracytoplasmic sperm injection and human microinjection.

PURPOSE: Intracytoplasmic sperm injection (ICSI) imparts physical stress on the oolemma of the oocyte and remains among the most technically demanding skills to master, with success rates related to experience and expertise. ICSI is also time-consuming and requires workflow management in the laboratory. This study presents a device designed to reduce the pressure on the oocyte during injection and investigates if this improves embryo development in a porcine model. The impact of this device on laboratory workflow was also assessed. METHODS: Porcine oocytes were matured in vitro and injected with porcine sperm by conventional ICSI (C-ICSI) or with microICSI, an ICSI dish that supports up to 20 oocytes housed individually in microwells created through microfabrication. Data collected included set-up time, time to align the polar body, time to perform the injection, the number of hand adjustments between controllers, and degree of invagination at injection. Developmental parameters measured included cleavage and day 6 blastocyst rates. Blastocysts were differentially stained to assess cell numbers of the inner cell mass and trophectoderm. A pilot study with human donated MII oocytes injected with beads was also performed. RESULTS: A significant increase in porcine blastocyst rate for microICSI compared to C-ICSI was observed, while cleavage rates and blastocyst cell numbers were comparable between treatments. Procedural efficiency of microinjection was significantly improved with microICSI compared to C-ICSI in both species. CONCLUSION: The microICSI device demonstrated significant developmental and procedural benefits for porcine ICSI. A pilot study suggests human ICSI should benefit equally.

Vertical transmission of maternal DNA through extracellular vesicles associates with altered embryo bioenergetics during the periconception period.

The transmission of DNA through extracellular vesicles (EVs) represents a novel genetic material transfer mechanism that may impact genome evolution and tumorigenesis. We aimed to investigate the potential for vertical DNA transmission within maternal endometrial EVs to the pre-implantation embryo and describe any effect on embryo bioenergetics. We discovered that the human endometrium secretes all three general subtypes of EV - apoptotic bodies (ABs), microvesicles (MVs), and exosomes (EXOs) - into the human endometrial fluid (EF) within the uterine cavity. EVs become uniformly secreted into the EF during the menstrual cycle, with the proportion of different EV populations remaining constant; however, MVs contain significantly higher levels of mitochondrial (mt)DNA than ABs or EXOs. During the window of implantation, MVs contain an eleven-fold higher level of mtDNA when compared to cells-of-origin within the receptive endometrium, which possesses a lower mtDNA content and displays the upregulated expression of mitophagy-related genes. Furthermore, we demonstrate the internalization of EV-derived nuclear-encoded (n)DNA/mtDNA by trophoblast cells of murine embryos, which associates with a reduction in mitochondrial respiration and ATP production. These findings suggest that the maternal endometrium suffers a reduction in mtDNA content during the preconceptional period, that nDNA/mtDNA become packaged into secreted EVs that the embryo uptakes, and that the transfer of DNA to the embryo within EVs occurs alongside the modulation of bioenergetics during implantation.

A rare case of mosaic Klinefelter syndrome in a 45-year-old man leading to successful live birth through ejaculated spermatozoa: a case report and literature review.

Background: Men diagnosed with Klinefelter syndrome (KS) commonly exhibit non-obstructive azoospermia or rarely having sperm in their ejaculate, rendering them traditionally considered sterile prior to the introduction of intracytoplasmic sperm injection (ICSI). The presence of mosaic KS may mask the classical phenotype, resulting in underdiagnosis throughout their lifetime. Surgical sperm retrieval through Microdissection Testicular Sperm Extraction (Micro-TESE) combined with ICSI has become the gold standard approach, maximizing reproductive outcomes in these individuals. However, it is noteworthy that approximately 7% of men with KS may exhibit sperm in their ejaculate, providing an opportunity for them to achieve biological parenthood through ICSI. Case Presentation: In this report, we present an exceptional case of a 45-year-old man with Mosaic KS and severe oligozoospermia who successfully achieved pregnancy utilizing ICSI with freshly ejaculated sperm. Remarkably, this case represents the oldest recorded instance of a man with Klinefelter syndrome fathering his own biological child using sperm derived from fresh ejaculate. Conclusion: Although this case is exceedingly rare, it underscores the critical importance of exhausting all possibilities to facilitate biological parenthood in men with KS before considering alternative options such as sperm donation or adoption. By recognizing the potential for successful conception using ejaculated sperm in this population, we can provide individuals with mosaic KS the opportunity to fulfill their desire for biological offspring.

Preimplantation embryo exposure to ketone bodies exerts sex-specific effects on mouse fetal and placental transcriptomes.

RESEARCH QUESTION: Does in vitro exposure of preimplantation mouse embryos to the ketone bodies ß-hydroxybutyrate (ßOHB) and acetoacetate (AcAc) impact post-transfer fetal and placental gene expression? DESIGN: Blastocysts cultured in vitro with or without 2 mmol/l ßOHB alone ('ßOHB') or combined with 0.8 mmol/l AcAc ('Keto') underwent embryo transfer. Transcriptional profiles of sexed placenta, liver and brain at gestational day 14.5 were examined via RNA sequencing and DAVID functional analysis. RESULTS: A sexually dimorphic response to in vitro ketone exposure was observed. Both ßOHB and Keto exposure down-regulated genes related to oxidative phosphorylation specifically in female liver. ßOHB down-regulated female placental steroid biosynthetic processes, while Keto treatment up-regulated genes relevant to blood vessel formation and cell migration in male placenta. Brain transcriptomes were minimally affected. X-linked genes and chromatin modifiers were identified as differentially expressed in both liver and placenta, alluding to a sex-specific regulatory mechanism. CONCLUSIONS: Transient preimplantation ketone exposure perturbs sex-specific fetal liver and placental gene expression, demonstrating a developmental programming effect that warrants future investigation of the postnatal metabolic health of male and female offspring.

Detrimental actions of obesity-associated advanced glycation end-products on endometrial epithelial cell proliferation are alleviated by antioxidants.

RESEARCH QUESTION: Advanced glycation end-products (AGE) are elevated in the uterine environment of obese infertile women. Can the detrimental effects of AGE on endometrial epithelial cells be mitigated with therapeutics, and recapitulated in a more physiologically relevant primary model (organoids)? DESIGN: Human endometrial epithelial cells (ECC-1) were exposed to AGE at concentrations physiologically representative of uterine fluid in lean or obese individuals, and three potential therapeutics: 25 nmol/l receptor for AGE (RAGE) antagonist FPS-ZM1, 100 µmol/l metformin, or a combination of antioxidants (10 µmol/l N-acetyl-l-cysteine, 10 µmol/l N-acetyl-l-carnitine and 5 µmol/l α-lipoic acid). Real-time cell analysis (xCELLigence, ACEA Biosciences) determined the rate of adhesion and proliferation. The proliferation of organoid-derived cells and secretion of cytokines from organoids was characterized in the presence of AGE (n = 5). The uterine fluid of women undergoing assisted reproduction was profiled for AGE-associated inflammatory markers (n = 77). RESULTS: ECC-1 proliferation was reduced by AGE from obese versus lean conditions and vehicle control (P = 0.04 and P < 0.001, respectively), and restored to a proliferation corresponding to lean conditions by antioxidants. AGE influenced organoid derived primary endometrial epithelial cell proliferation in a donor-dependent manner. AGE increased the organoid secretion of the proinflammatory cytokine CXCL16 (P = 0.006). Clinically, CXCL16 correlated positively to maternal body mass index (R = 0.264, P = 0.021) and intrauterine glucose concentration (R = 0.736, P < 0.0001). CONCLUSIONS: Physiologically relevant concentrations of AGE alter endometrial epithelial cell function. Antioxidants restore the rate of proliferation of AGE-treated endometrial epithelial (ECC-1) cells. Primary endometrial epithelial cells, cultured as organoids, demonstrate altered proliferation and CXCL16 secretion in the presence of AGE equimolar with the uterine fluid from obese individuals.

Nano-liter perfusion microfluidic device made entirely by two-photon polymerization for dynamic cell culture with easy cell recovery.

Polydimethylsiloxane (PDMS) has been the material of choice for microfluidic applications in cell biology for many years, with recent advances encompassing nano-scaffolds and surface modifications to enhance cell-surface interactions at nano-scale. However, PDMS has not previously been amenable to applications which require complex geometries in three dimensions for cell culture device fabrication in the absence of additional components. Further, PDMS microfluidic devices have limited capacity for cell retrieval following culture without severely compromising cell health. This study presents a designed and entirely 3D-printed microfluidic chip (8.8 mm × 8.2 mm × 3.6 mm) using two-photon polymerization (2PP). The 'nest' chip is composed of ten channels that deliver sub-microliter volume flowrates (to ~ 600 nL/min per channel) to 10 individual retrievable cell sample 'cradles' that interlock with the nest to create the microfluidic device. Computational fluid dynamics modelling predicted medium flow in the device, which was accurately validated by real-time microbead tracking. Functional capability of the device was assessed, and demonstrated the capability to deliver culture medium, dyes, and biological molecules to support cell growth, staining and cell phenotype changes, respectively. Therefore, 2PP 3D-printing provides the precision needed for nanoliter fluidic devices constructed from multiple interlocking parts for cell culture application.

Making and selecting the best embryo in the laboratory.

Over the past 4 decades our ability to maintain a viable human embryo in vitro has improved dramatically, leading to higher implantation rates. This has led to a notable shift to single blastocyst transfer and the ensuing elimination of high order multiple gestations. Future improvements to embryo culture systems will not only come from new improved innovative media formulations (such as the inclusion of antioxidants), but plausibly by moving away from static culture to more dynamic perfusion-based systems now made a reality owing to the breakthroughs in three-dimensional printing technology and micro fabrication. Such an approach has already made it feasible to create high resolution devices for intracytoplasmic sperm injection, culture, and cryopreservation, paving the way not only for improvements in outcomes but also automation of assisted reproductive technology. Although improvements in culture systems can lead to further increases in pregnancy outcomes, the ability to quantitate biomarkers of embryo health and viability will reduce time to pregnancy and decrease pregnancy loss. Currently artificial intelligence is being used to assess embryo development through image analysis, but we predict its power will be realized through the creation of selection algorithms based on the integration of information related to metabolic functions, cell-free DNA, and morphokinetics, thereby using vast amounts of different data types obtained for each embryo to predict outcomes. All of this will not only make assisted reproductive technology more effective, but it will also make it more cost effective, thereby increasing patient access to infertility treatment worldwide.

Acetoacetate and ß-hydroxybutyrate reduce mouse embryo viability via differential metabolic and epigenetic mechanisms.

RESEARCH QUESTION: Does the ketone acetoacetate (AcAc) alone, or combined with ß-hydroxybutyrate (ßOHB), impact mouse embryo development, metabolism, histone acetylation and viability? DESIGN: Pronucleate mouse oocytes were cultured in vitro in G1/G2 media supplemented with ketones (AcAc or AcAc + ßOHB) at concentrations representing those in maternal serum during pregnancy (0.04 mmol/l AcAc, 0.1 mmol/l ßOHB), standard diet consumption (0.1 mmol/l AcAc, 0.25 mmol/l ßOHB), ketogenic diet consumption (0.8 mmol/l AcAc, 2 mmol/l ßOHB) and diabetic ketoacidosis (2 mmol/l AcAc, 4 mmol/l ßOHB). Day 5 blastocysts were assessed for cell allocation, glucose metabolism and histone acetylation. Day 4 blastocysts exposed to 0.8 mmol/l AcAc + 2 mmol/l ßOHB were transferred to standard-fed recipient females, and E14.5 fetal and placental development assessed. RESULTS: Exposure to 2 mmol/l AcAc or 0.8 mmol/l AcAc + 2 mmol/l ßOHB did not impair blastocyst development, but significantly increased glucose consumption (P = 0.001 each), lowered glycolytic flux (P = 0.01, P < 0.001) and elevated trophectoderm (TE) histone 3 lysine 27 acetylation (H3K27ac; P < 0.001 each) compared with unexposed controls. Preimplantation AcAc + ßOHB exposure reduced post-implantation fetal development by 25% (P = 0.037), and delayed female-specific fetal limb development (P = 0.019) and estimated fetal age (P = 0.019) compared with controls. CONCLUSION: Preimplantation exposure to ketones affects underlying metabolism and histone acetylation in blastocysts that are associated with persistent, female-specific perturbations in fetal development. A periconceptional diet that elevates ketone concentrations may impair human embryonic viability.

Microfluidics facilitating the use of small extracellular vesicles in innovative approaches to male infertility.

Sperm are transcriptionally and translationally quiescent and, therefore, rely on the seminal plasma microenvironment for function, survival and fertilization of the oocyte in the oviduct. The male reproductive system influences sperm function via the binding and fusion of secreted epididymal (epididymosomes) and prostatic (prostasomes) small extracellular vesicles (S-EVs) that facilitate the transfer of proteins, lipids and nucleic acids to sperm. Seminal plasma S-EVs have important roles in sperm maturation, immune and oxidative stress protection, capacitation, fertilization and endometrial implantation and receptivity. Supplementing asthenozoospermic samples with normospermic-derived S-EVs can improve sperm motility and S-EV microRNAs can be used to predict non-obstructive azoospermia. Thus, S-EV influence on sperm physiology might have both therapeutic and diagnostic potential; however, the isolation of pure populations of S-EVs from bodily fluids with current conventional methods presents a substantial hurdle. Many conventional techniques lack accuracy, effectiveness, and practicality; yet microfluidic technology has the potential to simplify and improve S-EV isolation and detection.

Transient Polycomb activity represses developmental genes in growing oocytes.

BACKGROUND: Non-genetic disease inheritance and offspring phenotype are substantially influenced by germline epigenetic programming, including genomic imprinting. Loss of Polycomb Repressive Complex 2 (PRC2) function in oocytes causes non-genetically inherited effects on offspring, including embryonic growth restriction followed by post-natal offspring overgrowth. While PRC2-dependent non-canonical imprinting is likely to contribute, less is known about germline epigenetic programming of non-imprinted genes during oocyte growth. In addition, de novo germline mutations in genes encoding PRC2 lead to overgrowth syndromes in human patients, but the extent to which PRC2 activity is conserved in human oocytes is poorly understood. RESULTS: In this study, we identify a discrete period of early oocyte growth during which PRC2 is expressed in mouse growing oocytes. Deletion of Eed during this window led to the de-repression of 343 genes. A high proportion of these were developmental regulators, and the vast majority were not imprinted genes. Many of the de-repressed genes were also marked by the PRC2-dependent epigenetic modification histone 3 lysine 27 trimethylation (H3K27me3) in primary-secondary mouse oocytes, at a time concurrent with PRC2 expression. In addition, we found H3K27me3 was also enriched on many of these genes by the germinal vesicle (GV) stage in human oocytes, strongly indicating that this PRC2 function is conserved in the human germline. However, while the 343 genes were de-repressed in mouse oocytes lacking EED, they were not de-repressed in pre-implantation embryos and lost H3K27me3 during pre-implantation development. This implies that H3K27me3 is a transient feature that represses a wide range of genes in oocytes. CONCLUSIONS: Together, these data indicate that EED has spatially and temporally distinct functions in the female germline to repress a wide range of developmentally important genes and that this activity is conserved in the mouse and human germlines.

The role of timing in frozen embryo transfer.

The process of implantation is characterized by a complex cross-talk between the endometrium and the blastocyst, with the endometrium only being receptive to implantation during a transient window of implantation of approximately 2-3 days during the midsecretory phase. The timing of embryo transfer, including frozen embryo transfer, is therefore critical to the success of implantation. In this article, we discuss various elements that may guide the timing of frozen embryo transfer, including the role of endometrial characteristics such as thickness, days postovulation or length of progesterone administration, stage of the embryo, and the application of endometrial receptivity tests to guide personalized embryo transfer.

ß-hydroxybutyrate reduces blastocyst viability via trophectoderm-mediated metabolic aberrations in mice.

STUDY QUESTION: What is the effect of the ketone ß-hydroxybutyrate (ßOHB) on preimplantation mouse embryo development, metabolism, epigenetics and post-transfer viability? SUMMARY ANSWER: In vitro ßOHB exposure at ketogenic diet (KD)-relevant serum concentrations significantly impaired preimplantation mouse embryo development, induced aberrant glycolytic metabolism and reduced post-transfer fetal viability in a sex-specific manner. WHAT IS KNOWN ALREADY: A maternal KD in humans elevates gamete and offspring ßOHB exposure during conception and gestation, and in rodents is associated with an increased time to pregnancy, and altered offspring organogenesis, post-natal growth and behaviour, suggesting a developmental programming effect. In vitro exposure to ßOHB at supraphysiological concentrations (8-80 mM) perturbs preimplantation mouse embryo development. STUDY DESIGN, SIZE, DURATION: A mouse model of embryo development and viability was utilized for this laboratory-based study. Embryo culture media were supplemented with ßOHB at KD-relevant concentrations, and the developmental competence, physiology, epigenetic state and post-transfer viability of in vitro cultured ßOHB-exposed embryos was assessed. PARTICIPANTS/MATERIALS, SETTING, METHODS: Mouse embryos were cultured in vitro with or without ßOHB at concentrations representing serum levels during pregnancy (0.1 mM), standard diet consumption (0.25 mM), KD consumption (2 mM) and diabetic ketoacidosis (4 mM). The impact of ßOHB exposure on embryo development (blastocyst formation rate, morphokinetics and blastocyst total, inner cell mass and trophectoderm (TE) cell number), physiology (redox state, ßOHB metabolism, glycolytic metabolism), epigenetic state (histone 3 lysine 27 ß-hydroxybutyrylation, H3K27bhb) and post-transfer viability (implantation rate, fetal and placental development) was assessed. MAIN RESULTS AND THE ROLE OF CHANCE: All ßOHB concentrations tested slowed embryo development (P < 0.05), and ßOHB at KD-relevant serum levels (2 mM) delayed morphokinetic development, beginning at syngamy (P < 0.05). Compared with unexposed controls, ßOHB exposure reduced blastocyst total and TE cell number (≥0.25 mM; P < 0.05), reduced blastocyst glucose consumption (2 mM; P < 0.01) and increased lactate production (0.25 mM; P < 0.05) and glycolytic flux (0.25 and 2 mM; P < 0.01). Consumption of ßOHB by embryos, mediated via monocarboxylate transporters, was detected throughout preimplantation development. Supraphysiological (20 mM; P < 0.001), but not physiological (0.25-4 mM) ßOHB elevated H3K27bhb levels. Preimplantation ßOHB exposure at serum KD levels (2 mM) reduced post-transfer viability. Implantation and fetal development rates of ßOHB-treated embryos were 50% lower than controls (P < 0.05), and resultant fetuses had a shorter crown-rump length (P < 0.01) and placental diameter (P < 0.05). A strong sex-specific effect of ßOHB was detected, whereby female fetuses from ßOHB-treated embryos weighed less (P < 0.05), had a shorter crown-rump length (P < 0.05), and tended to have accelerated ear development (P < 0.08) compared with female control fetuses. LIMITATIONS, REASONS FOR CAUTION: This study only assessed embryo development, physiology and viability in a mouse model utilizing in vitro ßOHB exposure; the impact of in vivo exposure was not assessed. The concentrations of ßOHB utilized were modelled on blood/serum levels as the true oviduct and uterine concentrations are currently unknown. WIDER IMPLICATIONS OF THE FINDINGS: These findings indicate that the development, physiology and viability of mouse embryos is detrimentally impacted by preimplantation exposure to ßOHB within a physiological range. Maternal diets which increase ßOHB levels, such as a KD, may affect preimplantation embryo development and may therefore impair subsequent viability and long-term health. Consequently, our initial observations warrant follow-up studies in larger human populations. Furthermore, analysis of ßOHB concentrations within human and rodent oviduct and uterine fluid under different nutritional states is also required. STUDY FUNDING/COMPETING INTEREST(S): This work was funded by the University of Melbourne and the Norma Hilda Schuster (nee Swift) Scholarship. The authors have no conflicts of interest. TRIAL REGISTRATION NUMBER: N/A.

Antioxidant supplementation of mouse embryo culture or vitrification media support more in-vivo-like gene expression post-transfer.

RESEARCH QUESTION: What is the effect on mouse fetal gene expression of combined antioxidants (acetyl-L-carnitine, N-acetyl-L-cysteine and alpha-lipoic acid; A3) when used in culture media and vitrification/warming solutions? DESIGN: A laboratory-based analysis of an animal model. Embryo transfers were conducted on in-vivo-flushed blastocysts, or blastocysts cultured or vitrified with and without A3. Transcriptional profiles of E14.5 fetal liver and placental tissue in all groups were quantified using RNA-Seq and functional analyses (gene ontology [GO] biological processes and Kyoto Encyclopedia of Genes and Genomes [KEGG] pathway analysis). RESULTS: Both in-vitro culture in the presence of 20% oxygen and vitrification of blastocysts significantly perturbed fetal liver and placental gene expression. Notably, supplementation of in-vitro culture media or vitrification/warming solutions with A3 reduced the number of differentially expressed genes (DEG) and biological processes altered, establishing a more in-vivo-like gene expression profile, particularly within the E14.5 placenta. Specifically, A3 supplementation significantly reduced the expression of genes associated with pre-eclampsia and intrauterine growth restriction, along with genes involved in metabolism, cell senescence and cancer associated pathways. However, despite these improvements, several biological processes remained over-represented following both in-vitro culture and vitrification, even in the presence of A3. CONCLUSION: Both in-vitro culture in the presence of 20% oxygen and vitrification of blastocysts significantly perturbed fetal liver and placental gene expression, with the number of DEG greater following vitrification. Supplementation with A3 reduced the number of DEG and biological processes altered, establishing a more in-vivo-like gene expression profile, particularly in the placenta. Notably, A3 supplementation of in-vitro culture media significantly reduced the expression of genes associated with pre-eclampsia and intrauterine growth restriction.

Post-warming embryo morphology is associated with live birth: a cohort study of single vitrified-warmed blastocyst transfer cycles.

PURPOSE: This study aims to examine whether blastocyst morphology post-warming correlates with live birth. METHODS: In this cohort study, morphological characteristics post-warming were reviewed in all single vitrified-warmed blastocyst transfer cycles performed between November 2016 and May 2017. Immediately before transfer, the degree of blastocoel re-expansion was graded as A, fully expanded; B, partially expanded ≥ 50%; C, partially expanded < 50%; and D, collapsed. The degree of post-warming cell survival was graded on a scale of 50 to 100% and was then classified into 4 groups: very low 50-70%, low 71-80%, moderate 81-90%, and high 91-100%. RESULTS: Overall, 612 cycles were reviewed, of which 196 included PGT-A tested embryos. The live birth rate (LBR) increased from 11.4% in the collapsed blastocysts group to 38.9% in the post-warming full re-expansion group (p < 0.001) and from 6.5% for blastocysts with a very low cell survival rate to 34.7% for blastocysts with high cell survival rate (p = 0.001). LBR was 6.7% for blastocysts with the worst post-warming morphological characteristics, namely, collapsed with very low cell survival rate. On multivariate analyses, partial blastocyst re-expansion ≥ 50%, full re-expansion, and high cell survival rate remained significantly associated with live birth, after controlling for female age, pre-vitrification morphological grading, and PGT-A. A sub-analysis of cycles using PGT-A tested embryos showed similar results. CONCLUSION: Post-warming re-expansion and high cell survival rate are associated with higher LBR in euploid and untested blastocysts. However, embryos with poor post-warming morphology still demonstrate a considerable probability of live birth, and they should not be discarded.

A microenvironment of high lactate and low pH created by the blastocyst promotes endometrial receptivity and implantation.

RESEARCH QUESTION: Is the blastocyst's idiosyncratic metabolic production of lactate, and creation of a specialized microenvironment at the implatation site, an important mediator of maternal-fetal signalling to promote endometrial receptivity and implantation? DESIGN: Hormonally primed ECC-1 and Ishikawa cells were used to assess functional changes to the endometrial epithelium after exposure to lactic acid (LA), LA with neutralized pH (nLA) or acidic pH (pHL). Tight junction integrity (transepithelial resistance [TER]), cellular proliferation or changes to gene expression by RT-PCR were analysed. The effect of LA on Endometrial stromal cells decidualization and migratory capacity, and HUVEC endothelial tube formation and angiogenesis, were also assessed. RESULTS: Treatment of ECC-1 cells with 2.5 mM (P = 0.0037), 5 mM (P = 0.0044), 7.5 mM and 10 mM (P = 0.003) (P = 0.0021) LA significantly decreased the rate of cellular proliferation while TER was decreased with exposure to 2.5 mM LA (P = 0.024), 5 mM LA (P = 0.021) and 7.5 mM LA (P = 0.033). Exposure to nLA or pHL had no effect on proliferation or TER. Upregulation of GLUT4 (P = 0.002), GPR81 (P = 0.048), VEGF, SNAI1 (both P < 0.001) and RELA (P = 0.023) mRNA expression was observed after exposure of Ishikawa cells to combined LA plus pHL. Lactic acid increased the migratory capacity of decidualized stromal cells (P = 0.047) without changing the extent of decidualization. HUVEC tube formation was significantly increased by 5 mM LA exposure (P = 0.009). CONCLUSIONS: The identification of LA as an important mediator in the maternal-fetal dialogue underpinning implantation is supported. Further examination of the role of LA within the infertile or compromised endometrium could improve natural and assisted pregnancy success and needs further investigation.

'The way to improve ART outcomes is to introduce more technologies in the laboratory'.

To address the proposition that 'the way to improve ART outcomes is through the introduction of more technologies in the laboratory', it is prudent to first define what is considered to be improved outcomes. Evidently, this equates to an increase in the live birth rate but it should also include parameters such as time to pregnancy, cumulative pregnancy per oocyte retrieval and health of the resultant child. Furthermore, being able to maintain clinical results week in, week out through quality management also contributes to the overall success of a clinic, and hence can be considered an improved outcome. With regards to these outcomes, it is offered that not only does the introduction of several new technologies (defined here as instrumentation, techniques and enhanced computer utilization and analysis) have the potential to improve outcomes, but also some of them have the capacity to facilitate automation and standardization in the ART laboratory. Although the automation of procedures can be perceived as a justifiable goal itself, in this contribution the emphasis is on how new technologies could help more patients become parents of healthy children in the shortest possible time.