In vitro fertilization (IVF) and embryo culture in the human is a unique endeavor. Human assisted reproductive technology (ART) is practiced clinically to treat infertility. Due to the obvious ...ethical considerations of ART as applied to human medicine, only rarely is embryo culture undertaken for research purposes. As most IVF clinics are for profit businesses, a robust industry has developed to supply embryologists with quality control-tested media, equipment, and supplies necessary to support human IVF laboratory operation. Moreover, commercial items are preferred for efficiency and consistency, and strict quality control is required by accrediting organizations. As such, very little manual formulation or preparation of culture medium is typically required. Although human embryo culture is performed clinically, there is a surprising degree of variability in the details of the techniques used. In this chapter, we describe state-of-the-art procedures for gamete collection, in vitro fertilization, embryo culture, and embryo transfer that result in excellent blastocyst development and pregnancy rates for patients seeking treatment for infertility.
Exposure to environmental metals can cause nephrotoxicity. There is an international epidemic of chronic kidney disease of unknown cause (CKDu). Whether metal exposures contribute to kidney ...dysfunction in populations at risk for CKDu remains unresolved.
Urinary metals (arsenic, cadmium, nickel, and uranium) were examined in 222 sugarcane cutters in Guatemala at three time points over 1 year.
We explored the relationships between metal concentrations and markers of kidney function using multivariable linear mixed-effect models.
Arsenic, cadmium, and nickel were detected in the majority of the 340 urine samples and were generally within limits previously considered to be nonnephrotoxic. Nevertheless, higher urine cadmium was inversely associated with estimated glomerular filtration rate (eGFR) (β: -4.23, 95% confidence interval CI: -6.92, -1.54) and positively associated with neutrophil gelatinase-associated lipocalin (NGAL) (β: 2.92, 95% CI: 1.20, 4.64). Higher urine arsenic was also inversely associated with eGFR (β: -4.36, 95% CI: -7.07, -1.64).
Our findings suggest that exposures to metals, including cadmium and arsenic, might contribute to kidney toxicity seen in workers at risk for CKDu. These findings are consistent with the potential for metal nephrotoxicity at lower than expected levels in the setting of manual work in a very hot environment.
Introduction
Blood contacting medical devices, including rotary blood pumps, can cause shear‐induced blood damage that may lead to adverse effects in patients. Due in part to an inadequate ...understanding of how cell‐scale fluid mechanics impact red blood cell membrane deformation and damage, there is currently not a uniformly accepted engineering model for predicting blood damage caused by complex flow fields within ventricular assist devices (VADs).
Methods
We empirically investigated hemolysis in a magnetically levitated axial Couette flow device typical of a rotary VAD. The device is able to accurately control the shear rate and exposure time experienced by blood and to minimize the effects of other uncharacterized stresses. Using this device, we explored the effects of both hematocrit and plasma viscosity on shear‐induced hemolysis to characterize blood damage based on the viscosity‐independent shear rate, rather than on shear stress.
Results
Over a shear rate range of 20 000 – 80 000 1/s, the Index of Hemolysis (IH) was found to be dependent upon and well‐predicted by the shear rate alone. IH was independent of hematocrit, bulk viscosity, or the suspension media viscosity and less correlated to shear stress (MSE = 0.46–0.75) than to shear rate (MSE = 0.06–0.09).
Conclusion
This study recommends that future investigations of shear‐induced blood damage report findings with respect to the viscosity‐neutral term of shear rate, in addition to the bulk whole blood viscosity measured at an appropriate shear rate relevant to the flow conditions of the device.
We investigated hemolysis in a magnetically levitated axial Couette flow device typical of a rotary VAD over a controlled range of shear rate. The Index of Hemolysis was a function of shear rate, yet largely independent of hematocrit, bulk viscosity, or the suspension media viscosity. We recommended that future investigations of shear‐induced blood damage report findings with respect to the viscosity‐neutral term of shear rate, in addition to the bulk whole blood viscosity measured at an appropriate shear rate.
Maternal plasma 25-hydroxyvitamin D (25(OH)D) status and its association with pregnancy outcomes in malaria holoendemic regions of sub-Saharan Africa is poorly defined. We examined this association ...and any potential interaction with malaria and helminth infections in an ongoing pregnancy cohort study in Kenya. The association of maternal plasma 25(OH)D status with pregnancy outcomes and infant anthropometric measurements at birth was determined in a subset of women (
= 63). Binomial and linear regression analyses were used to examine associations between maternal plasma 25(OH)D and adverse pregnancy outcomes. Fifty-one percent of the women had insufficient (<75 nmol/L) and 21% had deficient (<50 nmol/L) plasma 25(OH)D concentration at enrollment. At birth, 74.4% of the infants had insufficient and 30% had deficient plasma 25(OH)D concentrations, measured in cord blood. Multivariate analysis controlling for maternal age and body mass index (BMI) at enrollment and gestational age at delivery found that deficient plasma 25(OH)D levels were associated with a four-fold higher risk of stunting in neonates (
= 0.04). These findings add to the existing literature about vitamin D and its association with linear growth in resource-limited settings, though randomized clinical trials are needed to establish causation.
Our objective was to determine the accuracy of identifying noncycling lactating dairy cows before the application of a timed artificial insemination (AI) protocol with or without progesterone ...supplementation via a controlled internal drug-release (CIDR) insert and 2 different timings of AI by using heatmount detectors and a single ovarian ultrasound examination. At 6 locations in the Midwest, 1,072 cows were enrolled in a Presynch protocol (2 injections of PGF₂α 14 d apart), with the second injection administered 14 d before initiating the Ovsynch protocol (injection of GnRH 7 d before and 48 h after PGF₂α injection, with timed AI at 0 or 24 h after the second GnRH injection). Heatmount detectors were applied to cows just before the first Presynch injection, assessed 14 d later at the second Presynch injection (replaced when activated or missing), and reassessed at initiation of the Ovsynch protocol. Ovaries were examined for the presence of a corpus luteum (CL) by ultrasound before the initiation of treatment. Treatments were assigned to cows based on the presence or absence of a CL detected by ultrasound: 1) no CL + no CIDR; 2) no CL + CIDR insert for 7 d; and 3) CL present. Further, alternate cows within the 3 treatments were assigned to be inseminated concurrent with the second GnRH injection of Ovsynch (0 h) or 24 h later. Pregnancy was diagnosed at 33 and 61 d after the second GnRH injection. By using low (<1 ng/mL) concentrations of progesterone in serum as the standard for noncycling status, heatmount detectors were activated on a large percentage of noncycling cows (>60%), whereas the single ultrasound examination incorrectly classified noncycling cows only 21% of the time. Conversely, cycling cows (progesterone >=1 ng/mL) were correctly identified 70 to 78% of the time by heatmount detectors, but 85 to 92% were correctly identified by ultrasound. Overall accuracy of heatmount detectors and ultrasound was 71 and 84%, respectively. Application of progesterone to cows without a CL at the time of the first injection of GnRH reduced the incidence of ovulation but increased the proportions of pregnancies per AI at d 33 or 61 compared with nontreated cows without a CL at the onset of the Ovsynch protocol. Percentages of cows pregnant and pregnancy survival did not differ for cows having a CL before treatment compared with those not having a CL and treated with progesterone. Compared with no response, when a follicle ovulated in response to the first GnRH injection, percentage of cows becoming pregnant after the timed AI increased from 33.3 to 41.6%. Timing of AI at 0 or 24 h after the second GnRH injection did not alter pregnancies per AI, but cows having luteal activity before treatment had improved pregnancies per AI compared with noncycling cows. We conclude that identifying noncycling cows by ultrasound was more accurate than by heatmount detectors. Subsequent progesterone treatment of previously cycling cows not having a CL at the onset of Ovsynch increased the proportion of pregnant cows, equal to that of cows having a CL but not treated with progesterone.
Abstract
STUDY QUESTION
What effect does maternal age have on the human oocyte's molecular response to in vitro oocyte maturation?
SUMMARY ANSWER
Although 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 ALREADY
Microarray 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, DURATION
The 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, METHODS
Patients 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 CHANCE
A 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 DATA
Raw data from this study can be accessed through GSE95477.
LIMITATIONS, REASONS FOR CAUTION
The 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 FINDINGS
Transcriptome 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.
Biofortification increases micronutrient content in staple crops through conventional breeding, agronomic methods, or genetic engineering. Bioaccessibility is a prerequisite for a nutrient to fulfill ...a biological function, e.g., to be bioavailable. The objective of this systematic review is to examine the bioavailability (and bioaccessibility as a proxy via in vitro and animal models) of the target micronutrients enriched in conventionally biofortified crops that have undergone post-harvest storage and/or processing, which has not been systematically reviewed previously, to our knowledge. We searched for articles indexed in MEDLINE, Agricola, AgEcon, and Center for Agriculture and Biosciences International databases, organizational websites, and hand-searched studies' reference lists to identify 18 studies reporting on bioaccessibility and 58 studies on bioavailability. Conventionally bred biofortified crops overall had higher bioaccessibility and bioavailability than their conventional counterparts, which generally provide more absorbed micronutrient on a fixed ration basis. However, these estimates depended on exact cultivar, processing method, context (crop measured alone or as part of a composite meal), and experimental method used. Measuring bioaccessibility and bioavailability of target micronutrients in biofortified and conventional foods is critical to optimize nutrient availability and absorption, ultimately to improve programs targeting micronutrient deficiency.
Biofortification is the process of increasing the concentrations and/or bioavailability of micronutrients in staple crops and has the potential to mitigate micronutrient deficiencies globally. ...Efficacy trials have demonstrated benefits of consuming biofortified crops (BFCs); and in this paper, we report on the results of a systematic review of biofortified crops effectiveness in real-world settings. We synthesized the evidence on biofortified crops consumption through four Impact Pathways: (1) purchased directly; (2) in informal settings; (3) in formal settings; or (4) in farmer households, from their own production. Twenty-five studies, covering Impact Pathway 1 (five studies), Impact Pathway 2 (three), Impact Pathway 3 (three), Impact Pathway 4 (21) were included. The review found evidence of an improvement in micronutrient status via Impact Pathway 4 (mainly in terms of vitamin A from orange sweet potato) in controlled interventions that involved the creation of demand, the extension of agriculture and promotion of marketing. In summary, evidence supports that biofortified crops can be part of food systems interventions to reduce micronutrient deficiencies in farmer households; ongoing and future research will help fully inform their potential along the other three Impact Pathways for scaling up.
Purpose
Estrogen is well-known for preparing uterine receptivity. However, its roles in regulating embryo development and implantation are unclear. Our objective was to characterize estrogen receptor ...1 (ESR1) in human and mouse embryos and determine the effect of estradiol (E
2
) supplementation on pre- and peri-implantation blastocyst development.
Methods
Mouse embryos, 8-cell through hatched blastocyst stages, and human embryonic days 5–7 blastocysts were stained for ESR1 and imaged using confocal microscopy. We then treated 8-cell mouse embryos with 8 nM E
2
during in vitro culture (IVC) and examined embryo morphokinetics, blastocyst development, and cell allocation into the inner cell mass (ICM) and trophectoderm (TE). Finally, we disrupted ESR1, using ICI 182,780, and evaluated peri-implantation development.
Results
ESR1 exhibits nuclear localization in early blastocysts followed by aggregation, predominantly in the TE of hatching and hatched blastocysts, in human and mouse embryos. During IVC, most E
2
was absorbed by the mineral oil, and no effect on embryo development was found. When IVC was performed without an oil overlay, embryos treated with E
2
exhibited increased blastocyst development and ICM:TE ratio. Additionally, embryos treated with ICI 182,780 had significantly decreased trophoblast outgrowth during extended embryo culture.
Conclusion
Similar ESR1 localization in mouse and human blastocysts suggests a conserved role in blastocyst development. These mechanisms may be underappreciated due to the use of mineral oil during conventional IVC. This work provides important context for how estrogenic toxicants may impact reproductive health and offers an avenue to further optimize human-assisted reproductive technology (ART) to treat infertility.
Abstract
STUDY QUESTION
Does a chemically defined maturation medium supplemented with FGF2, LIF, and IGF1 (FLI) improve in vitro maturation (IVM) of cumulus–oocyte complexes (COCs) obtained from ...children, adolescents, and young adults undergoing ovarian tissue cryopreservation (OTC)?
SUMMARY ANSWER
Although FLI supplementation did not increase the incidence of oocyte meiotic maturation during human IVM, it significantly improved quality outcomes, including increased cumulus cell expansion and mitogen-activated protein kinase (MAPK) expression as well as enhanced transzonal projection retraction.
WHAT IS KNOWN ALREADY
During OTC, COCs, and denuded oocytes from small antral follicles are released into the processing media. Recovery and IVM of these COCs is emerging as a complementary technique to maximize the fertility preservation potential of the tissue. However, the success of IVM is low, especially in the pediatric population. Supplementation of IVM medium with FLI quadruples the efficiency of pig production through improved oocyte maturation, but whether a similar benefit occurs in humans has not been investigated.
STUDY DESIGN, SIZE, DURATION
This study enrolled 75 participants between January 2018 and December 2021 undergoing clinical fertility preservation through the Fertility & Hormone Preservation & Restoration Program at the Ann & Robert H. Lurie Children’s Hospital of Chicago. Participants donated OTC media, accumulated during tissue processing, for research.
PARTICIPANTS/MATERIALS, SETTING, METHODS
Participants who underwent OTC and include a pediatric population that encompassed children, adolescents, and young adults ≤22 years old. All participant COCs and denuded oocytes were recovered from media following ovarian tissue processing. IVM was then performed in either a standard medium (oocyte maturation medium) or one supplemented with FLI (FGF2; 40 ng/ml, LIF; 20 ng/ml, and IGF1; 20 ng/ml). IVM outcomes included meiotic progression, cumulus cell expansion, transzonal projection retraction, and detection of MAPK protein expression.
MAIN RESULTS AND THE ROLE OF CHANCE
The median age of participants was 6.3 years, with 65% of them classified as prepubertal by Tanner staging. Approximately 60% of participants had been exposed to chemotherapy and/or radiation prior to OTC. On average 4.7 ± 1 COCs and/or denuded oocytes per participant were recovered from the OTC media. COCs (N = 41) and denuded oocytes (N = 29) were used for IVM (42 h) in a standard or FLI-supplemented maturation medium. The incidence of meiotic maturation was similar between cohorts (COCs: 25.0% vs 28.6% metaphase II arrested eggs in Control vs FLI; denuded oocytes: 0% vs 5.3% in Control vs FLI). However, cumulus cell expansion was 1.9-fold greater in COCs matured in FLI-containing medium relative to Controls and transzonal projection retraction was more pronounced (2.45 ± 0.50 vs 1.16 ± 0.78 projections in Control vs FLIat 16 h). Additionally, MAPK expression was significantly higher in cumulus cells obtained from COCs matured in FLI medium for 16–18 h (chemiluminescence corrected area 621,678 vs 2,019,575 a.u., P = 0.03).
LIMITATIONS, REASONS FOR CAUTION
Our samples are from human participants who exhibited heterogeneity with respect to age, diagnosis, and previous treatment history. Future studies with larger sample sizes, including adult participants, are warranted to determine the mechanism by which FLI induces MAPK expression and activation. Moreover, studies that evaluate the developmental competence of eggs derived from FLI treatment, including assessment of embryos as outcome measures, will be required prior to clinical translation.
WIDER IMPLICATIONS OF THE FINDINGS
FLI supplementation may have a conserved beneficial effect on IVM for children, adolescents, and young adults spanning the agricultural setting to clinical fertility preservation.
STUDY FUNDING/COMPETING INTEREST(S)
This work was supported by Department of Obstetrics and Gynecology startup funds (F.E.D.), Department of Surgery Faculty Practice Plan Grant and the Fertility & Hormone Preservation & Restoration Program at the Ann & Robert H. Lurie Children’s Hospital of Chicago (M.M.L. and E.E.R.). M.M.L. is a Gesualdo Foundation Research Scholar. Y.Y.’s research is supported by the internal research funds provided by Colorado Center of Reproductive Medicine. Y.Y., L.D.S., R.M.R., and R.S.P. have a patent pending for FLI. The remaining authors have no conflict of interest.
TRIAL REGISTRATION NUMBER
N/A.
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