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In vitro oocyte maturation is an assisted reproductive technology in which a meiotically immature oocyte (prophase I or germinal vesicle stage) is recovered from an antral follicle and matured in vitro prior to fertilization. This technology, although in widespread use in domestic livestock, is not typically implemented during human in vitro fertilization cycles. This review examines how in vitro oocyte maturation is currently used in the clinical setting, including the various ways in vitro oocyte maturation is defined in practice. The role of in vitro oocyte maturation in patient care and the major challenges for implementation are described. Efficiency and safety are critically explored. The role of in vitro oocyte maturation in oncofertility will also be discussed. Finally, the outlook for the future of clinical in vitro oocyte maturation is considered.

Recent studies in our laboratory have indicated that bovine embryos only use a small amount of the nutrients available to them in culture. Our objective was to evaluate the developmental and molecular response of bovine embryos when nutrient concentrations in the culture medium were significantly reduced. Following IVM and IVF, embryos were cultured in media containing 75, 50, and 25% (experiment 1) or 25, 12.5, and 6.25% (experiment 2) of the concentrations of nutrients (carbohydrates, amino acids, and vitamins) present in our control medium (100%). Blastocyst formation, hatching, and allocation of cells to the inner cell mass (ICM) and trophectoderm (TE) were evaluated on day 7. Although the number of TE cells was decreased (P < 0.05) when nutrient concentrations were ≤25% (73.8–124.1 cells), it was not until nutrient concentrations were reduced to 6.25% that blastocyst formation (18.3 ± 3.0%) and hatching (3.0 ± 1.3%) were inhibited (P < 0.05) compared to embryos cultured in the control medium (156.1 ± 14.1 cells, 40.0 ± 3.8%, 20.0 ± 3.1%, respectively). Inhibition of fatty acid oxidation (etomoxir) reduced (P < 0.05) blastocyst development, with more pronounced effects at lower nutrient concentrations (≤12.5%). Reducing nutrient concentrations was associated with increased activity of AMPK, decreased activity of mTOR, and altered abundance of transcripts for hexokinase 1 (HK1), carnitine palmitoyl transferase 2 (CPT2), lactate dehydrogenase A (LDHA), and pyruvate dehydrogenase kinase 1 (PDK1), consistent with an increase in glucose and fatty acid metabolism. Reduced nutrient conditions provide a unique perspective on embryo metabolism that may facilitate the optimization of culture media. Lay summary To support early embryo development in the first week after fertilisation, an appropriate mixture of nutrients (carbohydrates, amino acids, and vitamins) is needed in the culturing solution. However, refining these solutions to support optimal embryo health remains challenging. In this study, bovine (cow) embryos derived from abattoir material were used as a model for the development of other mammalian embryos, including humans. These embryos were cultured in the presence of 75, 50, 25, 12.5, or 6.25% of the nutrients present in control conditions (100%), which are similar to those reported for the fluids of the fallopian tubes and uterus. Embryo development was largely unaffected in the 75, 50, and 25% treatments, with some embryos developing in the presence of only 6.25% nutrients. Cow embryos are remarkably resilient to reduced concentrations of nutrients in their environment because they can utilize internal stores of fat as a source of energy.

To determine what patient and embryo characteristics are correlated with the developmental potential of the peri-implantation embryo.Retrospective study.Research laboratory.Six hundred fifty-one cryopreserved human blastocysts donated for research with informed patient consent.Not applicable.Blastocyst attachment to fibronectin-coated plates, trophectoderm outgrowth area, epiblast cell number, total cell number, human chorionic gonadotropin secretion.Patients' body mass index, age, follicle-stimulating hormone: luteinizing hormone ratio on menstrual cycle day 3, antral follicle count on menstrual cycle day 3, antimüllerian hormone level on menstrual cycle day 3, and blastocyst morphological grade were correlated with peri-implantation development outcomes. After controlling for good-quality morphological grades, blastocysts from patients of advanced maternal age developed fewer epiblast cells than blastocysts from younger patients.Extended embryo culture during the peri-implantation period mirrors several disparities in fertility treatment outcome that we see clinically, including those from patients with advanced maternal age, high body mass index, and low ovarian reserve and from embryos with lower-quality morphological grades. This model system may be useful by providing an alternative or more sensitive endpoint assessment in studying patient, clinical, or laboratory factors that may influence preimplantation embryo developmental potential.

Single-cell RNA sequencing of cells from cultured human blastocysts has enabled us to define the transcriptomic landscape of placental trophoblast (TB) that surrounds the epiblast and associated embryonic tissues during the enigmatic day 8 (D8) to D12 peri-implantation period before the villous placenta forms. We analyzed the transcriptomes of 3 early placental cell types, cytoTB (CTB), syncytioTB (STB), and migratoryTB (MTB), picked manually from cultured embryos dissociated with trypsin and were able to follow sublineages that emerged from proliferating CTB at the periphery of the conceptus. A unique form of CTB with some features of STB was detectable at D8, while mature STB was at its zenith at D10. A form of MTB with a mixed MTB/CTB phenotype arose around D10. By D12, STB generation was in decline, CTB had entered a new phase of proliferation, and mature MTB cells had begun to move from the main body of the conceptus. Notably, the MTB transcriptome at D12 indicated enrichment of transcripts associated with IFN signaling, migration, and invasion and up-regulation of HLA-C, HLA-E, and HLA-G. The STB, which is distinct from the STB of later villous STB, had a phenotype consistent with intense protein export and placental hormone production, as well as migration and invasion. The studies show that TB associated with human embryos is in rapid developmental flux during peri-implantation period when it must invade, signal robustly to the mother to ensure that the pregnancy continues, and make first contact with the maternal immune system.

Objective To assess the impact of shipment and storage of sperm, oocytes, and blastocysts in vapor phase nitrogen compared with static storage in liquid phase nitrogen. Design Prospective cohort-matched study. Setting Multiple in vitro fertilization laboratories in an in vitro fertilization network. Patient(s) Fifty-eight human embryos, 32 human oocytes, 15 units of bovine semen. Intervention(s) Vapor vs. liquid nitrogen. Main Outcome Measure(s) The postwarming survival of oocytes, sperm, and blastocysts, and the developmental potential of blastocysts during in vitro extended culture. Result(s) Custom-designed labware, for use with the TMRW platform, enables continuous temperature monitoring during shipment and/or storage in the vapor phase robotic storage system. The highest temperature recorded for specimens shipped to a domestic laboratory was −180.2 °C with a mean ± SD of −190.4 ± 0.5 °C during shipment and −181.1 ± 0.6 °C during storage. Likewise, specimens shipped internationally had a high of −180.2 °C with a mean ± SD of −193.5 ± 0.6 °C during shipment and −181.2 ± 0.7 °C during storage. Results from the extended culture assays have revealed no deleterious effect of shipment and storage in nitrogen vapor. The viability of mammalian gametes and embryos was equivalent between the vapor phase and liquid phase storage. Conclusion(s) The evaluated system did not have any deleterious effects on the postwarming survival of sperm, oocytes, and blastocysts. The postwarming developmental potential of human blastocysts during in vitro extended culture was unaffected by storage and handling in the vapor phase nitrogen TMRW platform when compared with static liquid phase nitrogen storage. Our results suggest that the vapor phase cryostorage platform is a safe system to handle and store reproductive specimens for human assisted reproductive technology.

Research Question: In the early stages of the COVID-19 pandemic, IVF clinics stopped the majority of patient treatment cycles to minimize the risk of disease transmission. The risk of SARS-CoV-2 viral exposure and potential cross contamination within the IVF lab remains largely unclear. To that end, the objective of this study was to examine follicular fluid (FF), culture media (M) and vitrification solution (VS) for SARS-CoV-2 in an IVF lab. Design: Prospective clinical study. All females undergoing transvaginal oocyte retrieval (TVOR) were required to have a negative SARS-CoV-2 RNA test 3-4 days prior to the procedure. Male partners were not tested. All cases utilized intracytoplasmic sperm injection (ICSI). The first tube of FF aspirated during oocyte retrieval, M drops following removal of embryos on day 5, and VS after blastocyst cryopreservation were analyzed for SARS-CoV-2 RNA. Results: In total, M from 61 patients, VS from 200 patients, and FF from 300 patients were analyzed. All samples were negative for SARS-CoV-2 viral RNA. Conclusion(s): With stringent safety protocols in place, including female patient testing and symptom-based screening of men, the presence of SARS-CoV-2 RNA was not detected in FF, M or VS. This work demonstrates the possibility of implementing a rapid laboratory screening assay for SARS-CoV-2 and has implications for safe laboratory operations, including cryostorage recommendations.

Summary Recent studies in our laboratory have indicated that bovine embryos only use a small amount of the nutrients available to them in culture. Our objective was to evaluate the developmental and molecular response of bovine embryos when nutrient concentrations in the culture medium were significantly reduced. Following IVM and IVF, embryos were cultured in media containing 75, 50, and 25% (experiment 1) or 25, 12.5, and 6.25% (experiment 2) of the concentrations of nutrients (carbohydrates, amino acids, and vitamins) present in our control medium (100%). Blastocyst formation, hatching, and allocation of cells to the inner cell mass (ICM) and trophectoderm (TE) were evaluated on day 7. Although the number of TE cells was decreased (P < 0.05) when nutrient concentrations were ≤25% (73.8–124.1 cells), it was not until nutrient concentrations were reduced to 6.25% that blastocyst formation (18.3 ± 3.0%) and hatching (3.0 ± 1.3%) were inhibited (P < 0.05) compared to embryos cultured in the control medium (156.1 ± 14.1 cells, 40.0 ± 3.8%, 20.0 ± 3.1%, respectively). Inhibition of fatty acid oxidation (etomoxir) reduced (P < 0.05) blastocyst development, with more pronounced effects at lower nutrient concentrations (≤12.5%). Reducing nutrient concentrations was associated with increased activity of AMPK, decreased activity of mTOR, and altered abundance of transcripts for hexokinase 1 (HK1), carnitine palmitoyl transferase 2 (CPT2), lactate dehydrogenase A (LDHA), and pyruvate dehydrogenase kinase 1 (PDK1), consistent with an increase in glucose and fatty acid metabolism. Reduced nutrient conditions provide a unique perspective on embryo metabolism that may facilitate the optimization of culture media. Lay summary To support early embryo development in the first week after fertilisation, an appropriate mixture of nutrients (carbohydrates, amino acids, and vitamins) is needed in the culturing solution. However, refining these solutions to support optimal embryo health remains challenging. In this study, bovine (cow) embryos derived from abattoir material were used as a model for the development of other mammalian embryos, including humans. These embryos were cultured in the presence of 75, 50, 25, 12.5, or 6.25% of the nutrients present in control conditions (100%), which are similar to those reported for the fluids of the fallopian tubes and uterus. Embryo development was largely unaffected in the 75, 50, and 25% treatments, with some embryos developing in the presence of only 6.25% nutrients. Cow embryos are remarkably resilient to reduced concentrations of nutrients in their environment because they can utilize internal stores of fat as a source of energy.

Further refinement of culture media is needed to improve the quality of embryos generated in vitro. Previous results from our laboratory demonstrated that uptake of nutrients by the embryo is significantly less than what is supplied in traditional culture media. Our objective was to determine the impact of reduced nutrient concentrations in culture medium on mouse embryo development, metabolism, and quality as a possible platform for next generation medium formulation. Concentrations of carbohydrates, amino acids, and vitamins could be reduced by 50% with no detrimental effects, but blastocyst development was impaired at 25% of standard nutrient provision (reduced nutrient medium; RN). Addition of pyruvate and L-lactate (+PL) to RN at 50% of standard concentrations restored blastocyst development, hatching, and cell number. In addition, blastocysts produced in RN + PL contained more ICM cells and ATP than blastocysts cultured in our control (100% nutrient) medium; however, metabolic activity was altered. Similarly, embryos produced in the RN medium with elevated (50% control) concentrations of pyruvate and lactate in the first step medium and EAA and Glu in the second step medium were competent to implant and develop into fetuses at a similar rate as embryos produced in the control medium. This novel approach to culture medium formulation could help define the optimal nutrient requirements of embryos in culture and provide a means of shifting metabolic activity towards the utilization of specific metabolic pathways that may be beneficial for embryo viability.

The objective of this work was to determine the role of mitochondria in the loss of oocyte quality with maternal aging. Our results show that mitochondrial DNA (mtDNA) copy number and function are reduced in eggs from aged mice after both in vivo and in vitro maturation. Higher incidences of spindle abnormalities were observed in old eggs. However, no correlation with egg ATP content was found. In vitro matured eggs from aged mice did not have a normal cortical distribution of active mitochondria and were subject to increased oxidative stress due to higher levels of reactive oxygen species and lower expression of glutamate-cysteine ligase, catalytic subunit (Gclc). Supplementation of antioxidants during in vitro maturation of old eggs mitigated this affect, resulting in increased mtDNA copy number and mitochondrial function, a mitochondria distribution pattern similar to young eggs, and improved chromosomal alignment. Eggs from women of advanced maternal age (AMA) had lower mitochondrial function than eggs from young women, although both age groups displayed a cortical distribution pattern of active mitochondria. In contrast to the mouse, human eggs from AMA women had higher mtDNA copy number than eggs from young women following in vitro maturation. In summary, oocytes of older females are more susceptible to perturbations in mitochondrial number and function, which are associated with increased spindle abnormalities and oxidative stress during in vitro maturation. These results demonstrate that oocyte mitochondria play a critical role in age-related infertility.

Carnivores are an interesting model for studies of embryonic amino acid metabolism and ammonium (NH4+) toxicity given the high-protein content of their diets. Our objectives were to examine concentration- and stage-specific effects of essential amino acids (EAA; 0×, 0.125×, 0.25×, 0.5×, or 1.0× the concentrations in Minimum Essential Medium) and NH4+ (0, 300, or 600 μM) on the development and metabolism of feline embryos. The presence of EAA, regardless of concentration, during days 3-7 of culture increased (P 0.01) the proportion of embryos that initiated hatching (14.3%) and the total number of cells per blastocyst (148.3 cells) compared to embryos cultured without EAA (0.0% and 113.2 ± 3.7 cells, respectively). The presence of EAA during days 1-3 (0.25×) and 3-7 (1.0×) of culture increased (P 0.01) the proportions of embryos that formed blastocysts (82.9 ± 4.2%) and initiated hatching (32.9 ± 5.2%), and the number of cells per blastocyst (247.9 ± 12.1 cells), compared to control embryos (60.0 ± 5.3%, 0.0%, 123.2 ± 8.1 cells, respectively). The presence of NH4+ in the medium did not affect (P 0.05) development of feline embryos. The addition of EAA or NH4+ during culture did not affect (P 0.05) the production of Gln by feline embryos, but decreased (P 0.05) production of Ala and increased (P 0.05) production of urea. Additional work is needed to determine if our observations are unique to feline embryos or reflect an adaptation to a high-protein diet that is conserved in other carnivores.

PURPOSE: The objective of this study was to evaluate the effects of multiple growth factors on the development of individually cultured murine embryos. METHODS: Embryos produced by in vitro fertilization using in vitro (IVM) or in vivo (IVO) matured oocytes from three strains of mice (CF1, Swiss Webster, B6D2F1) were cultured individually (10 μl) in the absence (control) or presence of growth factors (paf, epidermal growth factor [EGF], insulin-like growth factor 1 [IGF-1], and granulocyte-macrophage colony-stimulating factor [GM-CSF]). Blastocyst formation, hatching, and blastocyst cell numbers (trophectoderm, inner cell mass, and total) were evaluated on days 4 and 5 of culture. Post-hatching development of CF1 IVO embryos was also evaluated in vitro and in vivo. RESULTS: The presence of growth factors did not improve the proportion of embryos forming blastocysts or initiating hatching for any of the types of embryos tested. The only significant (P

Study question Can a pre-in vitro maturation (pre-IVM) medium containing signaling molecules rather than chemical/pharmaceutical agents, sustain meiotic arrest and improve developmental competence of in vitro matured oocytes in CF1 outbred mice? Summary answer A short 2 h period of pre-IVM prevents spontaneous meiotic resumption, improves mitochondria activity in subsequently matured oocytes, and increases developmental competence, pregnancy rate and implantation of resulting embryos. What is known already Spontaneous resumption of meiosis in vitro is detrimental for oocyte developmental competence. Pre-IVM systems that prevent spontaneous meiotic resumption with chemical/pharmaceutical agents are a promising approach to improving IVM oocyte competence; however, the success of these methods has proven to be inconsistent. Study design, size, duration This study consisted of a series of experiments using cumulus oocyte complexes (COC) derived from outbred mice following ovarian stimulation. The study was designed to examine if a novel, ligand/receptor-based pre-IVM treatment could sustain meiotic arrest in vitro and improve oocyte developmental competence, compared to control IVM. Two pre-IVM durations (2 h and 24 h) were evaluated, and the effect of the mitochondrial stimulator PQQ during 24 h pre-IVM was studied. Participants/materials, setting, methods Murine (outbred CF1) immature COC were cultured in vitro in the presence of C-type natriuretic peptide (CNP) (30 nM), estradiol (100 nM), FSH (1 × 10-4 IU/ml) and bone morphogenic protein 15 (BMP15) (100 ng/ml) for 2 h or 24 h prior to IVM. Meiotic status during pre-IVM and IVM was analyzed using orcein staining, and functionality of gap junction communication was confirmed using the functional gap junction inhibitor carbenoxolone (CBX). Oocytes exposed to pre-IVM treatment were compared to control oocytes collected on the same day from the same females and undergoing standard IVM. Developmental competence and embryo viability was assessed by oocyte mitochondrial activity and ATP concentration, in vitro embryo development following IVF and in vitro culture, blastocyst cell number and allocation, embryo morphokinetics, and embryo transfer. Differences were determined to be significant when P Main results and the role of chance Both a short (2 h) and long (24 h) pre-IVM period successfully prevented spontaneous resumption of meiosis. Moreover, gap junctions remained open during the pre-IVM period, as shown by the resumption of meiosis (95.9 ± 2.1%) in the presence of CBX during pre-IVM. A 2 h pre-IVM treatment improved blastocyst development after 96 h of culture per cleaved embryo compared to control (71.9 ± 7.4% versus 53.3 ± 6.2%, respectively), whereas a longer 24 h pre-IVM had no effect on development. A short 2 h period of pre-IVM increased mitochondrial activity in mature oocytes. On the contrary, mitochondrial activity was reduced in mature oocytes following 24 h of arrest and IVM. Treatment of arrested COC with pyrroloquinoline quinone (PQQ) during the 24 h pre-IVM period successfully maintained mitochondrial activity equal to control. However, PQQ was not able to improve blastocyst development compared to pre-IVM 24 h without PQQ. Moreover, ATP concentration in mature oocytes following pre-IVM and/or IVM, did not differ between treatments. A 2 h pre-IVM period prior to IVM improved pregnancy rate following transfer to recipient females. Implantation was also improved after transfer of embryos derived from oocytes arrested for either 2 h or 24 h prior to IVM, compared to control IVM derived embryos (41.9 ± 9%, 37.2 ± 9.5% and 17.2 ± 8.3%, respectively), although fetal development did not differ. Limitations, reasons for caution Slower meiotic resumption and enhanced mitochondrial activity likely contribute to improved developmental competence of oocytes exposed to pre-IVM for 2 h, but further experiments are required to identify specific mechanisms. Maintaining oocytes in meiotic arrest for 24 h with this approach could be a potential window to improve oocyte quality. However, an initial attempt to utilize this period of arrest to manipulate quality with PQQ, a mitochondrial stimulator, did not improve oocyte competence. Wider implications of the findings IVM could be an attractive clinical alternative to conventional IVF, with reduced time, cost and reliance on high doses of exogenous hormones to stimulate follicle growth, thus eliminating ovarian hyperstimulation syndrome (OHSS). Currently IVM is not widely used as it results in reduced embryo development and lower pregnancy outcomes compared to embryos produced from in vivo matured oocytes. Our approach to IVM, incorporating a ligand/receptor pre-IVM period, could improve human oocyte quality following IVM leading to routine adoption of this patient friendly technology. In addition, our methodology of pre-IVM containing signaling molecules rather than chemical/pharmaceutical agents may prove to be more consistent at improving oocyte quality than those focusing only on cAMP modulation with pharmacological agents. Finally, a reliable method of maintaining oocytes in meiotic arrest in vitro provides a novel window of opportunity in which the oocyte may be manipulated to address specific physiological deficiencies prior to meiotic resumption. Large scale data N/A. Study funding/competing interest(s) This study was supported by the Colorado Center for Reproductive Medicine (CCRM, Lone Tree, Colorado USA). We declare no conflict of interest.

The aim of this study is to evaluate the effect of a novel bovine cumulus oocyte complex (COC) shipping media designed to arrest meiotic resumption during transport on meiotic arrest, as well as meiotic resumption, subsequent embryonic development, and embryo quality. Bovine cumulus oocyte complexes were transported overnight from the collection facility to the laboratory. COCs were placed in control in vitro maturation (IVM) or in shipping arrest medium (SAM) containing multiple meiotic inhibitors, and then shipped to our laboratory. Upon arrival, meiotic status was assessed, control COCs were inseminated, and arrested COCs were matured and inseminated the next day. Embryonic development and quality were analyzed. When bovine COC arrived at the laboratory after overnight shipment (21 h) in SAM, the majority of oocytes remained at the GV stage (75.6 ± 2.9% GV). Arrested oocytes successfully resumed and completed meiosis during IVM after removal from SAM (96.8 ± 0.5% metaphase II compared to control 88.3 ± 5.0%). Moreover, the development of blastocysts per COC was not different from control (22.3 ± 2.4% for control and 18.7 ± 2.1% for SAM), nor was any difference detected in blastocyst quality as determined by cell number and allocation. Our study demonstrates that a physiological system incorporating cyclic adenosine monophosphate and cyclic guanosine monophosphate modulators can be used to maintain meiotic arrest followed by successful nuclear maturation and pre-implantation embryo development equal to control IVM-derived embryos. Our results offer promising insights for the development of pre-IVM media that may improve oocyte developmental competence in vitro.

Objective: SARS-CoV-2 entry in host cells requires the presence of angiotensin-converting enzyme 2 (ACE2) as the extracellular receptor, and the serine protease TMPRSS2 to cleave the viral spike protein for incorporation into the host cell. Basigin (BSG/CD147) may also act as an ACE2 independent receptor mechanism. The cysteine protease cathepsin-L (CTSL) may also cleave the viral spike proteins and facilitate cell entry. The objective of this study was to characterize the mRNA and protein expression of these cellular entry receptors and proteases in female reproductive cells to determine their susceptibility to SARS-CoV-2 infection. Design: Prospective Research Study. Materials and Methods: Materials and Methods: Oocytes (GV, MII), follicular cells (cumulus, CC;granulosa, GC) and embryos (1 cell, 1C;blastocyst, BL) were collected from a minimum of three different patients per sample type, with consent. Samples were analyzed for mRNA expression of ACE2, CD147, TMPRSS2, and CTSL genes relative to GAPDH using RT-qPCR. Primers were validated using human mixed tissue cDNA. Protein quantification was performed by immunoblotting using the Jess system (ProteinSimple) optimized to detect over 10 proteins/5-10 oocytes or embryos. Antibodies for ACE2, CD147, TMPRSS2, and CTSL proteins were validated and then used to determine protein abundance relative to total protein. Data were obtained from three independent biological replicates and analyzed using one-way ANOVA. Results: Results from q-PCR analysis revealed high (p

Objective: In the early stages of the COVID-19 pandemic, most IVF clinics stopped the majority of patient treatment cycles to minimize the risk of disease transmission When ASRM and other professional societies recommended resumption of treatments, procedures were put into place to ensure patient and staff safety However, the risk of SARS-CoV-2 viral exposure and potential cross contamination within the IVF laboratory remains largely unclear The objective of this study was to assess the true risk of exposure to SARS-CoV-2 in an active IVF laboratory when strict patient screening procedures are in place Design: Prospective analysis Materials and Methods: Prior to restarting IVF treatments, a COVID-19 safety protocol was implemented Patients and staff were required to wear masks, fill out a symptom-based questionnaire daily, have their temperature taken, and practice social distancing in patient waiting areas Each female patient undergoing transvaginal oocyte retrieval (TVOR) was required to have a negative SARS-CoV-2 RNA test 3-4 days prior to the procedure Male partners were not tested All cases examined utilized ICSI The first tube of follicular fluid aspirated during TVOR (FF), culture media drops following removal of embryos on day 5 (M), and vitrification solution (VS) after blastocyst cryopreservation were analyzed Self-inactivating replication incompetent lentivirus particles containing the single stranded viral RNA genome were immediately inoculated into each sample after collection as a positive control for viral RNA stability, prior to direct RNA isolation (M, VS) or sample concentration (FF) For FF, cell debris was removed by centrifugation and filtration (0 22 um) prior to concentration of virus particles with an Amicon filter RNA was isolated using the optimized QIAamp viral RNA minikit, RNA quantity and quality determined, and cDNA synthesized using SuperScript IV VILO master mix A multiplex TaqMan-based qPCR assay was developed for SARS-CoV-2 and lentivirus RNA (detection limit 5 SARS-CoV-2 copies/qPCR reaction and 50 viral copies/2 mL sample), and used to test all diagnostic samples SARS-Cov2 synthetic RNA and lentivirus RNA were used as an RT-qPCR positive control Samples with no amplification of lentivirus genome were removed from the analysis (false negative) Results: In total, culture medium from 30 patients, vitrification solution from 98 patients, and follicular fluid from 156 patients were analyzed All samples were negative for the presence of SARS-CoV-2 viral RNA Conclusions: With stringent safety protocols in place, including patient testing and use of ICSI, the presence of SARS-CoV-2 RNA can be avoided in the IVF laboratory Importantly, this study does not indicate that virus from an actively infected patient cannot be found in follicular fluid or make its way into the IVF lab However, it does provide reassurance that with proper patient testing and safety measures, cross-contamination of the virus between gametes and embryos (including within liquid nitrogen storage dewars), as well as exposure of embryologists, is minimal

The purpose of this review is to summarize what we know about preimplantation embryo metabolism, focusing on ruminant species, and to discuss how this knowledge informs our approach to culturing embryos in vitro. The important relationship between embryo metabolism and viability will be emphasized, and theories of metabolic networks in embryos presented. Methods that have historically been used to study embryo metabolism will be compared and contrasted to a new method of evaluating embryo metabolism; metabolomics. Finally, the advantages and disadvantages of using metabolomics technologies to study embryo metabolism will be critically evaluated. The application of metabolomics to assisted reproductive technologies, and specifically to embryo culture, will be highlighted. We conclude that use of metabolomics to study embryo physiology will enlighten our understanding of embryo metabolic pathways in the context of a complete media that enables good blastocyst production. This way of thinking about embryo metabolism as dynamic, complex and interrelated biochemical pathways, informed by metabolomics, will allow us to develop the next generation of embryo culture medium to support and manipulate metabolism to promote embryo viability, as well as to identify the most viable embryos for transfer.

The aim of this study is to compare the sensitivity of the standard one-cell mouse embryo assay (MEA) to that using in vitro-matured oocytes from hybrid and outbred mice. The study was done by culturing embryos in the presence or absence of two concentrations (0.0005 or 0.001 % v/v) of Triton X-100 (TX100). Embryonic development, blastocyst cell numbers (total and allocation to the trophectoderm [TE] and inner cell mass [ICM]), and blastocyst gene expression were evaluated. Neither concentration of TX100 affected (P > 0.05) cleavage, blastocyst development, or hatching in one-cell embryos from BDF1 mice. However, all cell number endpoints were reduced (P

The purpose of this study was to determine the effect of the protein stabilizer octanoic acid on blastocyst development, implantation, and fetal growth in a murine model. One-cell mouse embryos were collected and individually cultured in medium supplemented with recombinant human serum albumin for 96 h at 5 % oxygen in an EmbryoScope. Embryos were randomly allocated to four octanoic acid groups (0, 400, 800, or 1200 μM). Blastocyst development and cell cycle timings were calculated at 96 h of culture, and experiments were repeated in triplicate. Blastocysts were stained and fixed at 96 h for differential cell counts. Following 96 h of culture, blastocysts were transferred to recipients to determine implantation rates and fetal and placental weights. Blastocyst development, hatching rates, developmental kinetics, and total number of cells were negatively affected by octanoic acid at concentrations commonly used in human IVF. Implantation was not affected by octanoic acid but fetal and placental weights at 800 μM octanoic acid were increased relative to control. Octanoic acid, a standard additive to human protein supplements used in IVF, can have long-term negative effects on embryonic and fetal development. The use of octanoic acid for human embryo culture should be monitored and reduced.

Bidirectional somatic cell-oocyte signaling is essential to create a changing intrafollicular microenvironment that controls primordial follicle growth into a cohort of growing follicles, from which one antral follicle is selected to ovulate a healthy oocyte. Such intercellular communications allow the oocyte to determine its own fate by influencing the intrafollicular microenvironment, which in turn provides the necessary cellular functions for oocyte developmental competence, which is defined as the ability of the oocyte to complete meiosis and undergo fertilization, embryogenesis, and term development. These coordinated somatic cell-oocyte interactions attempt to balance cellular metabolism with energy requirements during folliculogenesis, including changing energy utilization during meiotic resumption. If these cellular mechanisms are perturbed by metabolic disease and/or maternal aging, molecular damage of the oocyte can alter macromolecules, induce mitochondrial mutations, and reduce adenosine triphosphate production, all of which can harm the oocyte. Recent technologies are now exploring transcriptional, translational, and post-translational events within the human follicle with the goal of identifying biomarkers that reliably predict oocyte quality in the clinical setting.

It was reported that mitochondrial DNA (mtDNA) was significantly increased in aneuploid human embryos compared to euploid embryos and was also associated with maternal age. In this study, we further established the mouse model of mtDNA quantitation in reproductive samples based on whole-genome amplification (WGA) and next-generation sequencing (NGS). WGA followed by NGS-based mtDNA quantitation was first performed on 6 single- and 100-cell samples from a tumor-derived mouse cell line, which was exposed to ethidium bromide to reduce mtDNA content. The relative mtDNA content was normalized to nuclear DNA. This method was then applied to mouse reproductive samples, including 40 pairs of oocytes and polar bodies from 8 CD-1 female mice of advanced reproductive age and 171 blastocysts derived via in vitro maturation (IVM) or in vivo maturation (IVO) from young (6–9 weeks) and reproductively aged (13.5 months) female CF-1 mice. Exposure to ethidium bromide for 3 and 6 days decreased mtDNA levels in both the single- and 100-cell samples as expected. Results demonstrated that the first polar body contained an average of 0.9% of mtDNA relative to oocytes. Compared to the cells in blastocysts, oocytes contained about 180 times as much mtDNA per cell. mtDNA levels were compared among blastocysts from reproductively young and old female mice that had either been produced by IVM or IVO. Cells in blastocysts from younger mice contained significantly lower amounts of mtDNA compared to aged mice (P

To determine the composition of commercially available protein supplements for embryo culture media and test if differences in protein supplement composition are biologically relevant in a murine model.Amino acid, organic acid, ion and metal content were determined for 6 protein supplements: recombinant human albumin (AlbIX), human serum albumin (HSA and Buminate), and three complex protein supplements (SSS, SPS, LGPS). To determine if differences in the composition of these supplements are biologically relevant, mouse one-cell embryos were collected and cultured for 120 hours in each protein supplement in Global media at 5 and 20 % oxygen in an EmbryoScope time-lapse incubator. The compositions of six protein supplements were analyzed for concentrations of 39 individual amino acids, organic acids, ions and elements. Blastocyst development and cell cycle timings were calculated at 96-hours of culture and the experiments were repeated in triplicate. Blastocyst gene expression was analyzed.Recombinant albumin had the fewest undefined components , the lowest concentration of elements detected, and resulted in high blastocyst development in both 5 and 20 % oxygen. Buminate, LGPS and SPS had high levels of transition metals whereas SSS had high concentrations of amino acids. Pre-compaction mouse embryo development was delayed relative to embryos in AlbIX for all supplements and blastocyst formation was reduced in Buminate, SPS and SSS.The composition of protein supplements are variable, consisting of previously undescribed components. High concentrations of pro-oxidant transition metals were most notable. Blastocyst development was protein dependent and showed an interaction with oxygen concentration and pro-oxidant supplements.

Study questionWhat effect does maternal age have on the human oocyte's molecular response to in vitro oocyte maturation?Summary answerAlthough polyadenylated transcript abundance is similar between young and advanced maternal age (AMA) germinal vesicle (GV) oocytes, metaphase II (MII) oocytes exhibit a divergent transcriptome resulting from a differential response to in vitro oocyte maturation.What is known alreadyMicroarray studies considering maternal age or maturation stage have shown that either of these factors will affect oocyte polyadenylated transcript abundance in human oocytes. However, studies considering both human oocyte age and multiple stages simultaneously are limited to a single study that examined transcript levels for two genes by qPCR. Thus, polyadenylated RNA sequencing (RNA-Seq) could provide novel insight into age-associated aberrations in gene expression in GV and MII oocytes.Study design, size, durationThe effect of maternal age (longitudinal analysis) on polyadenylated transcript abundance at different stages was analyzed by examining single GV and single in vitro matured MII oocytes derived from five young (YNG; < 30 years; average age 26.8; range 20-29) and five advanced maternal age (AMA; ≥40 years; average age 41.6 years; range 40-43 years) patients. Thus, a total of 10 YNG (5 GV and 5 MII) and 10 AMA (5 GV and 5 MII) oocytes were individually processed for RNA-Seq analysis.Participants/materials, settings, methodsPatients undergoing infertility treatment at the Colorado Center for Reproductive Medicine (Lone Tree, CO, USA) underwent ovarian stimulation with FSH and received hCG for final follicular maturation prior to ultrasound guided oocyte retrieval. Unused GV oocytes obtained at retrieval were donated for transcriptome analysis. Single oocytes were stored (at -80°C in PicoPure RNA Extraction Buffer; Thermo Fisher Scientific, USA) immediately upon verification of immaturity or after undergoing in vitro oocyte maturation (24 h incubation), representing GV and MII samples, respectively. After isolating RNA and generating single oocyte RNA-Seq libraries (SMARTer Ultra Low Input RNA HV kit; Clontech, USA), Illumina sequencing (100 bp paired-end reads on HiSeq 2500) and bioinformatics analysis (CLC Genomics Workbench, DESeq2, weighted gene correlation network analysis (WGCNA), Ingenuity Pathway Analysis) were performed.Main results and the role of chanceA total of 12 770 genes were determined to be expressed in human oocytes (reads per kilobase per million mapped reads (RPKM) > 0.4 in at least three of five replicates for a minimum of one sample type). Differential gene expression analysis between YNG and AMA oocytes (within stage) identified 1 and 255 genes that significantly differed (adjusted P < 0.1 and log2 fold change >1) in polyadenylated transcript abundance for GV and MII oocytes, respectively. These genes included CDK1, NLRP5 and PRDX1, which have been reported to affect oocyte developmental potential. Despite the similarity in transcript abundance between GV oocytes irrespective of age, divergent expression patterns emerged during oocyte maturation. These age-specific differentially expressed genes were enriched (FDR < 0.05) for functions and pathways associated with mitochondria, cell cycle and cytoskeleton. Gene modules generated by WGCNA (based on gene expression) and patient traits related to oocyte quality (e.g. age and blastocyst development) were correlated (P < 0.05) and enriched (FDR < 0.05) for functions and pathways associated with oocyte maturation.Large scale dataRaw data from this study can be accessed through GSE95477.Limitations, reasons for cautionThe human oocytes used in the current study were obtained from patients with varying causes of infertility (e.g. decreased oocyte quality and oocyte quality-independent factors), possibly affecting oocyte gene expression. Oocytes in this study were retrieved at the GV stage following hCG administration and the MII oocytes were derived by IVM of patient oocytes. Although the approach has the benefit of identifying intrinsic differences between samples, it may not be completely representative of in vivo matured oocytes.Wider implications of the findingsTranscriptome profiles of YNG and AMA oocytes, particularly at the MII stage, suggest that aberrant transcript abundance may contribute to the age-associated decline in fertility.Study funding/competing interest(s)J.M.R. was supported by an Austin Eugene Lyons Fellowship awarded by the University of California, Davis. The Eunice Kennedy Shriver National Institute of Child Health and Human Development of the National Institutes of Health (awarded to P.J.R.; R01HD070044) and the Fertility Laboratories of Colorado partly supported the research presented in this manuscript.

Advanced reproductive age is unequivocally associated with increased aneuploidy in human oocytes, which contributes to infertility, miscarriages, and birth defects. The frequency of meiotic chromosome segregation errors in oocytes derived from reproductively aged mice appears to be similar to that observed in humans, but a limitation of this important model system is our inability to accurately identify chromosome-specific aneuploidy. Here we report the validation and application of a new low-pass whole-genome sequencing approach to comprehensively screen chromosome aneuploidy in individual mouse oocytes and blastocysts. First, we validated this approach by using single mouse embryonic fibroblasts engineered to have stable trisomy 16. We further validated this method by identifying reciprocal chromosome segregation errors in the products of meiosis I (gamete and polar body) in oocytes from reproductively aged mice. Finally, we applied this technology to investigate the incidence of aneuploidy in blastocysts derived from in vitro- and in vivo-matured oocytes in both young and reproductively aged mice. Using this next generation sequencing approach, we quantitatively assessed meiotic and mitotic segregation errors at the single chromosome level, distinguished between errors due to premature separation of sister chromatids and classical nondisjunction of homologous chromosomes, and quantified mitochondrial DNA (mtDNA) segregation in individual cells. This whole-genome sequencing technique, therefore, greatly improves the utility of the mouse model system for the study of aneuploidy and is a powerful quantitative tool with which to examine the molecular underpinnings of mammalian gamete and early embryo chromosome segregation in the context of reproductive aging and beyond.

In this essay, we propose that embryos express a metabolic phenotype necessarily different from that of differentiated somatic cells and more like that of rapidly proliferating cancer cells. This metabolic adaptation, known as the Warburg Effect, supports rapid cell proliferation. One of the hallmarks of the Warburg Effect is that pyruvate is directed away from the tri-carboxylic acid cycle and metabolized to lactate, resulting in a buildup of glycolytic intermediates. Although this is a comparatively inefficient way to generate ATP, this adaptation allows the cell to meet other critical metabolic requirements, including biomass production and redox regulation. Thus, utilization of WE gives proliferating cells a selective growth advantage. This model represents a completely new understanding of embryo metabolism in the context of a broad, interconnected network of metabolic mechanisms that influence viability, versus the current dogma of carbohydrate metabolism via oxidative phosphorylation. A more complete understanding of embryo metabolism is critical to better support embryo viability in vitro, and to avoid forcing embryos to adapt to suboptimal culture conditions at a significant cost to future growth and development.