Metabolic regulation of preimplantation embryo development in vivo and in vitro: Molecular mechanisms and insights.

Understanding of embryonic development has led to the clinical application of Assisted Reproductive technologies (ART), with the resulting birth of millions of children. Recent developments in metabolomics, proteomics, and transcriptomics have brought to light new insights into embryonic growth dynamics, with implications spanning reproductive medicine, stem cell research, and regenerative medicine. The review explores the key metabolic processes and molecular pathways active during preimplantation embryo development, including PI3K-Akt, mTOR, AMPK, Wnt/ß-catenin, TGF-ß, Notch and Jak-Stat signaling pathways. We focused on analyzing the differences occurring in vitro as opposed to in vivo development and we discussed significant physiological and clinical implications.

Outcomes of Very Preterm Infants Conceived with Assisted Reproductive Technologies.

OBJECTIVE: To compare mortality and early respiratory outcomes of very preterm infants conceived via assisted reproductive technology (ART) vs spontaneously. STUDY DESIGN: We identified inborn infants (July 2014-July 2019) with gestational age <32 weeks (n = 439); 54 cases were ART conceived. Spontaneously conceived controls (n = 103) were matched by multiple gestation status and gestational age. Primary outcome was 1-year mortality. Secondary outcomes were receipt of respiratory support and supplemental oxygen at 7 and 28 days and 36 weeks of postmenstrual age. We evaluated the association between conception method and outcomes by logistic regression, with adjustment for sociodemographic status. RESULTS: Women who conceived via ART had increased rates of prepregnancy and gestational diabetes, and no differences in rates of hypertensive disorders. Infant 1-year mortality was not different by mode of conception (ART 11.8% vs spontaneous 7.1%, P = .49). Infants conceived by ART were less likely to receive respiratory support or supplemental oxygen at all time points, but this relationship only reached significance for receipt of oxygen at 28 days (ART 20.8% vs spontaneous 39.0%, P = .03); this remained true after adjustment for race/ethnicity and socioeconomic index. CONCLUSIONS: When controlling for gestational age and multiple gestation status, very preterm infants conceived following ART had similar outcomes as those conceived spontaneously.

Paternal developmental toxicant exposure is associated with epigenetic modulation of sperm and placental Pgr and Igf2 in a mouse model.

Preterm birth (PTB), parturition prior to 37 weeks' gestation, is the leading cause of neonatal mortality. The causes of spontaneous PTB are poorly understood; however, recent studies suggest that this condition may arise as a consequence of the parental fetal environment. Specifically, we previously demonstrated that developmental exposure of male mice (F1 animals) to the environmental endocrine disruptor 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) was associated with reduced sperm quantity/quality in adulthood and control female partners frequently delivered preterm. Reproductive defects persisted in the F2 and F3 descendants, and spontaneous PTB was common. Reproductive changes in the F3 males, the first generation without direct TCDD exposure, suggest the occurrence of epigenetic alterations in the sperm, which have the potential to impact placental development. Herein, we conducted an epigenetic microarray analysis of control and F1 male-derived placentae, which identified 2171 differentially methylated regions, including the progesterone receptor (Pgr) and insulin-like growth factor (Igf2). To assess if Pgr and Igf2 DNA methylation changes were present in sperm and persist in future generations, we assessed methylation and expression of these genes in F1/F3 sperm and F3-derived placentae. Although alterations in methylation and gene expression were observed, in most tissues, only Pgr reached statistical significance. Despite the modest gene expression changes in Igf2, offspring of F1 and F3 males consistently exhibited IUGR. Taken together, our data indicate that paternal developmental TCDD exposure is associated with transgenerational placental dysfunction, suggesting epigenetic modifications within the sperm have occurred. An evaluation of additional genes and alternative epigenetic mechanisms is warranted.

LIMK1 regulates human trophoblast invasion/differentiation and is down-regulated in preeclampsia.

Successful human pregnancy requires extensive invasion of maternal uterine tissues by the placenta. Invasive extravillous trophoblasts derived from cytotrophoblast progenitors remodel maternal arterioles to promote blood flow to the placenta. In the pregnancy complication preeclampsia, extravillous trophoblasts invasion and vessel remodeling are frequently impaired, likely contributing to fetal underperfusion and maternal hypertension. We recently demonstrated in mouse trophoblast stem cells that hypoxia-inducible factor-2 (HIF-2)-dependent Lim domain kinase 1 (LIMK1) expression regulates invasive trophoblast differentiation by modulating the trophoblast cytoskeleton. Interestingly, in humans, LIMK1 activity promotes tumor cell invasion by modulating actin and microtubule integrity, as well as by modulating matrix metalloprotease processing. Here, we tested whether HIF-2α and LIMK1 expression patterns suggested similar roles in the human placenta. We found that LIMK1 immunoreactivity mirrored HIF-2α in the human placenta in utero and that LIMK1 activity regulated human cytotrophoblast cytoskeletal integrity, matrix metallopeptidase-9 secretion, invasion, and differentiation in vitro. Importantly, we also found that LIMK1 levels are frequently diminished in the preeclampsia setting in vivo. Our results therefore validate the use of mouse trophoblast stem cells as a discovery platform for human placentation disorders and suggest that LIMK1 activity helps promote human placental development in utero.

A role for the chemokine receptor CCR6 in mammalian sperm motility and chemotaxis.

Although recent evidence indicates that several chemokines and defensins, well-known as inflammatory mediators, are expressed in the male and female reproductive tracts, the location and functional significance of chemokine networks in sperm physiology and sperm reproductive tract interactions are poorly understood. To address this deficiency in our knowledge, we examined the expression and function in sperm of CCR6, a receptor common to several chemoattractant peptides, and screened several reproductive tract fluids for the presence of specific ligands. CCR6 protein is present in mouse and human sperm and mainly localized in the sperm tail with other minor patterns in sperm from mice (neck and acrosomal region) and men (neck and midpiece regions). As expected from the protein immunoblotting and immunofluorescence results, mouse Ccr6 mRNA is expressed in the testis. Furthermore, the Defb29 mRNA encoding the CCR6 ligand, ß-defensin DEFB29, is expressed at high levels in the epididymis. As determined by protein chip analysis, several chemokines (including some that act through CCR6, such as CCL20/MIP-3α (formerly macrophage inflammatory protein 3α) and protein hormones were present in human follicular fluid, endometrial secretions, and seminal plasma. In functional chemotaxis assays, capacitated human sperm exhibited a directional movement towards CCL20, and displayed modifications in motility parameters. Our data indicate that chemokine ligand/receptor interactions in the male and female genital tracts promote sperm motility and chemotaxis under non-inflammatory conditions. Therefore, some of the physiological reactions mediated by CCR6 ligands in male reproduction extend beyond a pro-inflammatory response and might find application in clinical reproduction and/or contraception.

In vitro fertilization affects growth and glucose metabolism in a sex-specific manner in an outbred mouse model.

The preimplantation period is a time of reprogramming that may be vulnerable to disruption. This question has wide clinical relevance since the number of children conceived by in vitro fertilization (IVF) is rising. To examine this question, outbred mice (CF1 × B6D2F1) conceived by IVF and cultured using Whitten medium and 20% O2 (IVFWM group, less optimal) or K simplex optimized medium with amino acids and 5% O2 (IVFKAA group, more optimal and similar to conditions used in human IVF) were studied postnatally. We found that flushed blastocysts transferred to recipient mice provided the best control group (FB group), as this accounted for the effects of superovulation, embryo transfer, and litter size. We observed that many physiological parameters were normal. Reassuringly, IVFKAA offspring did not differ significantly from FB offspring. However, male IVFWM mice (but not females) were larger during the first 19 wk of life and exhibited glucose intolerance. Male IVFWM mice also showed enlarged left heart despite normal blood pressure. Expression of candidate imprinted genes (H19, Igf2, and Slc38a4) in multiple adult tissues did not show differences among the groups; only Slc38a4 was down-regulated following IVF (in both culture conditions) in female adipose tissue. These studies demonstrate that adult metabolism is affected by the type of conditions encountered during the preimplantation stage. Further, the postnatal growth trajectory and glucose homeostasis following ex vivo manipulation may be sexual dimorphic. Future work on the long-term effects of IVF offspring should focus on glucose metabolism and the cardiovascular system.

The effect of fertility treatment and socioeconomic status on neonatal and post-neonatal mortality in the United States.

OBJECTIVE: To determine the association between fertility treatment, socioeconomic status (SES), and neonatal and post-neonatal mortality. STUDY DESIGN: Retrospective cohort study of all births (19,350,344) and infant deaths from 2014-2018 in the United States. The exposure was mode of conception-spontaneous vs fertility treatment. The outcome was neonatal (<28d), and post-neonatal (28d-1y) mortality. Multivariable logistic models were stratified by SES. RESULT: The fertility treatment group had statistically significantly higher odds of neonatal mortality (high SES OR 1.59; CI [1.5, 1.68], low SES OR 2.11; CI [1.79, 2.48]) and lower odds of post-neonatal mortality (high SES OR 0.87, CI [0.76, 0.996], low SES OR 0.6, CI [0.38, 0.95]). SES significantly modified the effect of ART/NIFT on neonatal and post-neonatal mortality. CONCLUSIONS: Fertility treatment is associated with higher neonatal and lower post-neonatal mortality and SES modifies this effect. Socioeconomic policies and support for vulnerable families may help reduce rates of infant mortality.

Metabolomic Profile of Children Conceived With Medically Assisted Technologies.

CONTEXT: Assisted reproductive technologies (ART) and non-in vitro fertilization fertility treatments (NIFT) are treatments for infertility. These technologies may have long-term health effects in children such as increased hypertension, glucose intolerance, and hypertriglyceridemia. Few studies have compared children born following ART and NIFT to those conceived spontaneously by subfertile couples. OBJECTIVE: This work aimed to describe metabolic differences in children conceived by ART and NIFT compared to children conceived spontaneously by infertile couples. METHODS: Children conceived by parent(s) receiving infertility care at the University of California, San Francisco, between 2000 and 2017 were invited to participate in the Developmental Epidemiological Study of Children born through Reproductive Technology (DESCRT). Serum metabolomic analyses were conducted using samples from 143 enrolled children (age range 4-12 years, 43% female) conceived using NIFT or ART (with fresh or frozen embryos with and without intracytoplasmic sperm injection [ICSI]) and children conceived spontaneously by subfertile couples. Principal component analysis and multivariable regression were used to compare the distribution of metabolites between groups. RESULTS: There was no separation in metabolites based on treatment or sex. NIFT-conceived children showed no differences compared to spontaneously conceived controls. Only spontaneously conceived children had different metabolomics profiles from children conceived from fresh ART, frozen ART, and all ICSI. Pantoate and propionylglycine levels were elevated in fresh ART compared to the spontaneous group (P < .001). Propionylglycine levels were elevated in the ICSI (both fresh and frozen) vs the spontaneous group (P < .001). Finally, 5-oxoproline levels were decreased in frozen ART compared to the spontaneous group (P < .001). CONCLUSION: NIFT-conceived children did not show any metabolic differences compared with spontaneously conceived children. The metabolic differences between ART-conceived children and children conceived spontaneously were small but unlikely to be clinically significant but should be examined in future studies.

Fertility treatment increases the risk of preterm birth independent of multiple gestations.

Objective: To investigate the complex interplay between fertility treatment, multiple gestations, and prematurity. Design: Retrospective cohort study linking the national Center for Disease Control and Prevention infant birth and death data from 2014 to 2018. Setting: National database from Center of Disease Control and Prevention. Patients: In total, 19,454,155 live-born infants with gestational ages 22-44 weeks, 114,645 infants born using non IVF fertility treatment (NIFT), and 179,960 via assisted reproductive technology (ART). Intervention: Noninvasive fertility treatment or ART vs. spontaneously conceived pregnancies. Main Outcome Measures: The main outcome assessed was prematurity. Formal mediation analysis was conducted to calculate the percentage mediated by multiple gestations. Results: Newborns born using NIFT or ART compared with those with no fertility treatment had a higher incidence of multiple gestation (no fertility treatment = 3.0%; NIFT = 24.7%; ART = 32.7%; P<.001) and prematurity (no fertility treatment = 11.2%; NIFT = 23.4%; ART = 28.4%; P<.001). Mediation analysis demonstrates that 76.8% (95% confidence interval [CI], 75.2%-78.1%) of the effect of NIFT on prematurity was mediated through multiple gestations. Similarly, 71.2% (95% CI, 70.8%-72.7%) of the effect of ART on prematurity is mediated through multiple gestation. However, the direct effect of NIFT on prematurity is 20.4% (95% CI, 19.0%-22.0%). The direct effect of ART was 24.7% (95% CI, 23.7%-25.6%). Conclusion: A significant proportion of prematurity associated with fertility treatment is mediated by the treatment itself, independent of multiple gestations.

Murine blastocysts generated by in vitro fertilization show increased Warburg metabolism and altered lactate production.

In vitro fertilization (IVF) has resulted in the birth of over 8 million children. Although most IVF-conceived children are healthy, several studies suggest an increased risk of altered growth rate, cardiovascular dysfunction, and glucose intolerance in this population compared to naturally conceived children. However, a clear understanding of how embryonic metabolism is affected by culture condition and how embryos reprogram their metabolism is unknown. Here, we studied oxidative stress and metabolic alteration in blastocysts conceived by natural mating or by IVF and cultured in physiologic (5%) or atmospheric (20%) oxygen. We found that IVF-generated blastocysts manifest increased reactive oxygen species, oxidative damage to DNA/lipid/proteins, and reduction in glutathione. Metabolic analysis revealed IVF-generated blastocysts display decreased mitochondria respiration and increased glycolytic activity suggestive of enhanced Warburg metabolism. These findings were corroborated by altered intracellular and extracellular pH and increased intracellular lactate levels in IVF-generated embryos. Comprehensive proteomic analysis and targeted immunofluorescence showed reduction of lactate dehydrogenase-B and monocarboxylate transporter 1, enzymes involved in lactate metabolism. Importantly, these enzymes remained downregulated in the tissues of adult IVF-conceived mice, suggesting that metabolic alterations in IVF-generated embryos may result in alteration in lactate metabolism. These findings suggest that alterations in lactate metabolism are a likely mechanism involved in genomic reprogramming and could be involved in the developmental origin of health and disease.

The effect of follicular fluid hormones on oocyte recovery after ovarian stimulation: FSH level predicts oocyte recovery.

BACKGROUND: Ovarian stimulation for assisted reproductive technology (ART) overcomes the physiologic process to develop a single dominant follicle. However, following stimulation, egg recovery rates are not 100%. The objective of this study is to determine if the follicular fluid hormonal environment is associated with oocyte recovery. METHODS: This is a prospective study involving patients undergoing ART by standard ovarian stimulation protocols at an urban academic medical center. A total of 143 follicular fluid aspirates were collected from 80 patients. Concentrations of FSH, hCG, estradiol, progesterone, testosterone and prolactin were determined. A multivariable regression analysis was used to investigate the relationship between the follicular fluid hormones and oocyte recovery. RESULTS: Intrafollicular FSH was significantly associated with oocyte recovery after adjustment for hCG (Adjusted odds ratio (AOR) = 1.21, 95%CI 1.03-1.42). The hCG concentration alone, in the range tested, did not impact the odds of oocyte recovery (AOR = 0.99, 95%CI 0.93-1.07). Estradiol was significantly associated with oocyte recovery (AOR = 0.98, 95% CI 0.96-0.99). After adjustment for progesterone, the strength of association between FSH and oocyte recovery increased (AOR = 1.84, 95%CI 1.45-2.34). CONCLUSION: The relationship between FSH and oocyte recovery is significant and appears to work through mechanisms independent of the sex hormones. FSH may be important for the physiologic event of separation of the cumulus-oocyte complex from the follicle wall, thereby influencing oocyte recovery. Current methods for inducing the final stages of oocyte maturation, with hCG administration alone, may not be optimal. Modifications of treatment protocols utilizing additional FSH may enhance oocyte recovery.

Outcomes of pulmonary vascular disease in infants conceived with non-IVF fertility treatment and assisted reproductive technologies at 1 year of age.

BACKGROUND: Assisted reproductive technologies (ARTs) have been associated with the development of endothelial dysfunction. OBJECTIVE: To determine potential differences in outcomes associated with pulmonary vascular disease in infants born to mothers receiving any infertility treatment including ART and non-IVF fertility treatments (NIFTs). DESIGN/METHODS: The sample was derived from an administrative database containing detailed information on infant and maternal characteristics for live-born infants in California (2007-2012) with gestational age (GA) 22 to 44 weeks. Cases were defined as infants with ICD-9 code for pulmonary vascular disease (PVD) and records for ART/NIFT. Controls were randomly selected at a 1:4 ratio. The primary outcome was 1-year mortality. Crude and adjusted odds ratio (OR) with 95% confidence interval (CI) were calculated. RESULTS: We identified 159 cases and 636 controls. Mothers that utilized ART/NIFT were older, to be of the Caucasian race, to have pre-eclampsia, private insurance, and education >12 years (P < .001). Cases compared to controls were more premature, had lower birth weights, and were more often the product of a multiple gestation pregnancy (P < .001). Cases had a higher 1-year mortality (18.2% vs 9.1%; OR: 2.2; 95% CI: 1.4, 3.6), more severe PVD (86.2% vs 72.3%; OR: 2.4; 95% CI: 1.5, 3.9), and a longer hospital stay (66.7 ± 73.0 vs 32.5 ± 47.2 days; P < .001) than controls. However, when adjusting for GA these differences become statistically insignificant. CONCLUSION: Children born following ART/NIFT with PVD had increased mortality compared to infants with PVD but without ART/NIFT. The primary driver of this relationship is prematurity.

Advanced Paternal Age Affects Sperm Count and Anogenital Distance in Mouse Offspring.

In Western society, couples increasingly delay parenthood until later in life. Overall, studies have focused on the reproductive performance of older parents or the impact of advanced maternal age on pregnancy outcomes, but few studies have examined how advanced paternal age (APA) affects offspring health. The aim of this study was to investigate the impact of increasing paternal age on offspring reproductive performance and long-term metabolic health in a mouse model. Here, the same adult B6D2F1/J male mice were mated at 4, 12, and 18 months of age with 6- to 10-week-old naturally cycling CF1 females to generate 3 offspring cohorts conceived at increasing paternal ages PA4, PA12, and PA18. The offspring resulting from mating the same fathers at different ages (n = 20 per age; 10 males and 10 females) were maintained up to 20 weeks of age and morphometric parameters, growth curve, and glucose tolerance were measured. We found that increasing paternal age was associated with a trend toward longer time to conception. Litter sizes were not significantly different. Reassuringly, metabolic parameters and growth curve were not different in the 3 cohorts of offspring. Most importantly, increased paternal age (PA4 vs PA18) was associated with a statistically significant decrease in sperm concentration, sperm motility, and anogenital distance in offspring. These changes raise concerns about the potential impact of APA on the reproductive fitness in males of the next generation.

150 years of congenital adrenal hyperplasia: translation and commentary of De Crecchio's classic paper from 1865.

In 1865, Luigi De Crecchio, a Neapolitan pathologist, published a detailed autopsy description of Giuseppe Marzo, who lived as a man, had nearly-normal appearing male external genitalia, female internal reproductive organs, and massively enlarged adrenals. This report is widely cited as the first report of non-salt-losing congenital adrenal hyperplasia (CAH), but a complete English translation has not been available. Via interlibrary loan, we obtained the original volume containing De Crecchio's paper. The complete 39-page publication was translated by two reproductive endocrinologists (L.D.P. and P.F.R.) who are native speakers of Italian. A pediatric endocrinologist conversant with CAH (W.L.M.) summarizes and comments on De Crecchio's observations. Anatomically, the external genitalia were characterized by labio-scrotal fusion and a 10-cm curved phallus with hypospadias. Internally, the ovaries, tubes, and uterus were hypoplastic but otherwise normal, except that the uterus inserted into a utricle. The adrenals were massively enlarged, but this observation was dismissed as unimportant. De Crecchio's exposition of Marzo's life shows many of the issues affecting patients today: family ill-ease regarding genital ambiguity at birth, social pressure following reversed sex assignment in childhood, adult embarassment about genital appearance, difficulties with a legal sex assignment on a birth certificate, and substantial efforts to exhibit maleness to self and associates. De Crecchio was an astute observer who provides insight into both nineteenth-century endocrinology and continuing twenty-first-century difficulties in the care of patients with disordered sex development.

Unwind and transcribe: chromatin reprogramming in the early mammalian embryo.

Within the first few days of life, the unipotent gametic genomes are rapidly reprogrammed to support emergence of pluripotent cells in the early mammalian embryo. It is now appreciated that this crucial stage of development involves dramatic changes to chromatin at multiple levels, such as DNA methylation, histone modifications, histone mobility, and higher-order chromatin organization. Technological advances are beginning to allow genome-wide views of this chromatin reprogramming, and provide new approaches to functionally dissect its regulation. Here we review recent insights into the dynamic chromatin environment of the early mouse embryo. New data challenge long-held assumptions, for example, with regards to the asymmetry of DNA methylation of the parental genomes or the onset of functional zygotic genome activation. We discuss how impaired chromatin reprogramming can lead to early embryonic lethality, but might also have delayed effects that only manifest later in embryogenesis or postnatally, potentially influencing the propensity for adult-onset diseases.

HIGD1A Regulates Oxygen Consumption, ROS Production, and AMPK Activity during Glucose Deprivation to Modulate Cell Survival and Tumor Growth.

Hypoxia-inducible gene domain family member 1A (HIGD1A) is a survival factor induced by hypoxia-inducible factor 1 (HIF-1). HIF-1 regulates many responses to oxygen deprivation, but viable cells within hypoxic perinecrotic solid tumor regions frequently lack HIF-1α. HIGD1A is induced in these HIF-deficient extreme environments and interacts with the mitochondrial electron transport chain to repress oxygen consumption, enhance AMPK activity, and lower cellular ROS levels. Importantly, HIGD1A decreases tumor growth but promotes tumor cell survival in vivo. The human Higd1a gene is located on chromosome 3p22.1, where many tumor suppressor genes reside. Consistent with this, the Higd1a gene promoter is differentially methylated in human cancers, preventing its hypoxic induction. However, when hypoxic tumor cells are confronted with glucose deprivation, DNA methyltransferase activity is inhibited, enabling HIGD1A expression, metabolic adaptation, and possible dormancy induction. Our findings therefore reveal important new roles for this family of mitochondrial proteins in cancer biology.

Primary psoas abscess complicating a normal vaginal delivery.

BACKGROUND: Psoas abscess is a rare and potentially dangerous complication of normal delivery. CASE: We describe a case of primary psoas abscess after normal vaginal delivery. A young woman presented with fever, left back pain, left lower abdominal pain, and hip pain starting on postpartum day 2. Computed tomography demonstrated a large retroperitoneal collection. Aspiration drainage of the abscess under computed tomography guidance isolated Streptococcus viridans, which responded to antibiotic therapy and percutaneous drainage with complete resolution. CONCLUSION: A high index of suspicion is necessary for diagnosis of psoas abscess, which should be considered in postpartum patients with pyrexia, back and hip pain, and a normal neurologic examination. Computed tomography is effective for diagnosis and allows percutaneous drainage of the abscess.