Spatially resolved transcriptomic profiling of ovarian aging in mice.

Ovarian aging precedes that of any other mammalian organ and is the primary cause of female age-related infertility. The biological mechanisms responsible for ovarian aging remain unclear. Previous studies have been limited by their use of bulk RNA-sequencing, which masks the dynamic and heterogeneous nature of the ovary. In this study, we spatially resolved the transcriptomic landscape of ovaries from young and aged outbred mice. In total, we defined eight main ovarian cell populations, all of which were characterized by significant transcriptomic changes between young and aged samples. Further sub-cluster analysis revealed separate transcriptomes for distinct granulosa cell populations found in young versus aged mice, in addition to an oocyte sub-cluster population completely absent from aged mouse ovaries. This study provides a new perspective on mammalian ovarian aging using spatial transcriptomics to achieve deeper understanding of the localization and cell-population-specific mechanisms underlying age-related fertility decline.

Euploid day 7 blastocysts of infertility patients with only slow embryo development have reduced implantation potential.

RESEARCH QUESTION: What is the reproductive potential of embryos that achieve blastulation on day 7 followed by preimplantation genetic testing for aneuploidies (PGT-A) for infertility patients with slow embryo development? DESIGN: This was a retrospective cohort study in a private IVF clinic of consecutive female infertility patients (n = 2966) aged 24-48 (36.3 ± 3.8) years who underwent frozen embryo transfer (FET) of a single euploid blastocyst. RESULTS: The women underwent single euploid FET of an embryo that achieved blastulation on day 5 (n = 1880), day 6 (n = 986) or day 7 (n = 100). Day 7 embryos resulted in lower implantation and live birth rates compared with both day 5 and day 6 embryos (P < 0.001). The day 5, day 6 and day 7 groups had 68.5%, 55.2% and 36.0% live birth rates, respectively. The day 7 group was older than the day 5 group (P < 0.001); comparing age-matched cohorts, the day 7 group still had lower implantation and live birth rates (P < 0.0001 and P < 0.001, respectively). Embryo grade was unrelated to live birth rates. Day 7 embryos of expansion grade 5 or 6 or trophectoderm grade A were more likely to be euploid compared with expansion grade 3 or trophectoderm grade B. CONCLUSIONS: Euploid day 7 embryos represented reduced implantation potential, even when controlling for maternal age. Of all day 7 embryos that underwent PGT-A, euploidy was associated with expansion grade 5 or 6 and trophectoderm grade A. These results can help providers manage patient expectations in cases where infertile women have slow embryo development.

Increased Systemic Antioxidant Power Ameliorates the Aging-Related Reduction in Oocyte Competence in Mice.

Ovarian aging is associated with elevated oxidative stress and diminished oocyte developmental competence. We aimed to determine the impact of systemic antioxidant treatment in aged mice. Female outbred CF-1 mice were aged for 9 months prior to an 8-week 45 mg Euterpe oleracea (açaí) daily supplement. The açaí treatment induced a threefold increase in serum antioxidant power (FRAP) compared to both young and aged mice (p < 0.0001). Compared to young mice, aged mice had fewer oocytes and reduced blastocyst development (p < 0.0001); açaí did not affect the oocyte numbers, but improved blastocyst formation (p < 0.05). Additionally, açaí alleviated the aging-related decrease in implantation potential (p < 0.01). The aged mice showed evidence of elevated ovarian ER stress (increased whole-ovary PDIA4 expression, granulosa cell and oocyte GRP78 expression, and oocyte PDIA4 protein), reduced oocyte mitochondrial quality (higher PRKN activation and mitochondrial DNA oxidative damage), and dysregulated uterine glandular epithelium. Antioxidant intervention was sufficient to lessen these effects of ovarian aging, likely in part by the upregulation of NRF2. We conclude that açaí treatment is a promising strategy to improve ER and mitochondrial function in the ovaries, thereby ameliorating the decreased oocyte competence that occurs with ovarian aging.

Liquid chromatography-tandem mass spectrometry reveals an active response to DNA damage in human spermatozoa.

OBJECTIVE: To investigate how endogenously elevated DNA fragmentation alters the human sperm proteome, and whether this fragmentation contributes to genomic deletions. DESIGN: Research study. SETTING: Commercial fertility clinic. PATIENT(S): Men with low (0%-4%, n = 7) or high (≥16%, n = 6) sperm DNA fragmentation, as assessed by terminal deoxynucleotidyl transferase dUTP nick end labeling assay. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Global sperm proteome, single-nucleotide polymorphism genotyping array. RESULT(S): A total of 78 significantly differentially abundant proteins (30 decreased, 48 increased) were observed in control vs. high DNA damage samples. DNA damage resulted in robust proteomic responses, including markers of oxidative stress and apoptosis, DNA damage repair proteins, and transcription/translation and protein turnover machinery. Several key sperm functional proteins were significantly decreased in ejaculates with high DNA damage. We were unable to substantiate a link between increased DNA fragmentation and genomic deletions in human spermatozoa. CONCLUSION(S): Developing human spermatozoa initiate an active transcriptional response to endogenous DNA damage, which manifests as alterations in the sperm proteome.

Antioxidant Intervention Attenuates Aging-Related Changes in the Murine Ovary and Oocyte.

Advanced maternal age (AMA) is associated with reduced fertility due in part to diminished ovarian follicle quantity, inferior oocyte quality, chromosome aneuploidy, and lower implantation rates. Ovarian aging is accompanied by increased oxidative stress and blunted antioxidant signaling, such that antioxidant intervention could improve reproductive potential. The first aim of this study was to determine the molecular effects of antioxidant intervention in the ovaries and oocytes of aged mice, utilizing a supplement containing only naturally occurring açaí (Euterpe oleracea) with an oxygen radical absorbance capacity of 208,628 µmol Trolox equivalent (TE)/100 g indicating high antioxidant activity. Nine month old female CF-1 mice were administered 80 mg/day antioxidants (n = 12) or standard diet (n = 12) for 12 weeks. In the ovary, antioxidant treatment upregulated ß-adrenergic signaling, downregulated apoptosis and proinflammatory signaling, and variably affected cell growth and antioxidant pathways (p < 0.05). Exogenous antioxidants also increased the oocyte expression of antioxidant genes GPX1, SOD2, and GSR (p < 0.05). A feasibility analysis was then conducted on female AMA infertility patients as a proof-of-principle investigation. Patients (n = 121; <45 years old) consented to receiving 600 mg antioxidants three times daily for ≥8 weeks preceding infertility treatment. Preliminary results indicate promising outcomes for AMA patients, warranting further investigation.

AMP-activated protein kinase activator AICAR attenuates hypoxia-induced murine fetal growth restriction in part by improving uterine artery blood flow.

KEY POINTS: Pregnancy at high altitude is associated with a greater incidence of fetal growth restriction due, in part, to lesser uterine artery blood flow. AMP-activated protein kinase (AMPK) activation vasodilates arteries and may increase uterine artery blood flow. In this study, pharmacological activation of AMPK by the drug AICAR improved fetal growth and elevated uterine artery blood flow. These results suggest that AMPK activation is a potential strategy for improving fetal growth and raising uterine artery blood flow in pregnancy, which may be important in pregnancy disorders characterized by uteroplacental ischaemia and/or fetal hypoxia. ABSTRACT: Uteroplacental hypoxia is associated with pregnancy disorders such as intrauterine growth restriction and preeclampsia, which are characterized by uteroplacental ischaemia and/or fetal hypoxia. Activation of AMP-activated protein kinase (AMPK) results in vasodilatation and is therefore a potential therapeutic strategy for restoring uteroplacental perfusion in pregnancy disorders. In this study, C57Bl/6 mice were treated with subcutaneous pellets containing vehicle, the AMPK activator AICAR (200 mg kg-1 day-1 ), or the AMPK inhibitor Compound C (20 mg kg-1 day-1 ) beginning on gestational day 13.5, and were exposed to hypoxia starting on gestational day 14.5 that induced intrauterine growth restriction. Pharmacological AMPK activation by AICAR partially prevented hypoxia-induced fetal growth restriction (P < 0.01), due in part to increased uterine artery blood flow (P < 0.0001). The proportion of total cardiac output flowing through the uterine artery was increased with AICAR in hypoxic mice (P < 0.001), suggesting that the vasodilator effect of AICAR was selective for the uterine circulation. Further, pharmacological inhibition of AMPK with Compound C reduced uterine artery diameter and increased uterine artery contractility in normoxic mice, providing evidence that physiological levels of AMPK activation are necessary for vasodilatation in healthy pregnancy. Two-way ANOVA analyses indicated that hypoxia reduced AMPK activation in the uterine artery and placenta, and AICAR increased AMPK activation in these tissues compared to vehicle. These findings provide support for further investigation into the utility of pharmacological AMPK activation for treatment of fetal growth restriction.

Peroxisome proliferator-activated receptor gamma blunts endothelin-1-mediated contraction of the uterine artery in a murine model of high-altitude pregnancy.

The environmental hypoxia of high altitude (HA) increases the incidence of intrauterine growth restriction (IUGR) approximately threefold. The peroxisome proliferator-activated receptor γ (PPAR-γ), a ligand-activated nuclear receptor that promotes vasorelaxation by increasing nitric oxide and downregulating endothelin-1 (ET-1) production, has been implicated in IUGR. Based on our prior work indicating that pharmacologic activation of the PPARγ pathway protects against hypoxia-associated IUGR, we used an experimental murine model to determine whether such effects may be attributed to vasodilatory effects in the uteroplacental circulation. Using wire myography, ex vivo vasoreactivity studies were conducted in uterine arteries (UtA) isolated from pregnant mice exposed to hypoxia or normoxia from gestational day 14.5 to 18.5. Exposure to troglitazone, a high-affinity PPARγ agonist-induced vasorelaxation in UtA preconstricted with phenylephrine, with HA-UtA showing increased sensitivity. Troglitazone blunted ET-1-induced contraction of UtA in hypoxic and normoxic dams equivalently. Immunohistological analysis revealed enhanced staining for ET-1 receptors in the placental labyrinthine zone in hypoxic compared to normoxic dams. Our results suggest that pharmacologic PPAR-γ activation, via its vasoactive properties, may protect the fetal growth under hypoxic conditions by improving uteroplacental perfusion and thereby justify further investigation into PPARγ as a therapeutic target for IUGR in pregnancies complicated by hypoxia.

Increased uterine artery blood flow in hypoxic murine pregnancy is not sufficient to prevent fetal growth restriction†.

Incomplete maternal vascular responses to pregnancy contribute to pregnancy complications including intrauterine growth restriction (IUGR) and preeclampsia. We aimed to characterize maternal vascular dysfunction in a murine model of fetal growth restriction as an approach toward identifying targetable pathways for improving pregnancy outcomes. We utilized a murine model of late-gestation hypoxia-induced IUGR that reduced E18.5 fetal weight by 34%. Contrary to our hypothesis, uterine artery blood flow as measured in vivo by Doppler ultrasound was increased in mice housed under hypobaric hypoxia (385 mmHg; 5500 m) vs normoxia (760 mmHg; 0 m). Using wire myography, uterine arteries isolated from hypoxic mice had similar vasodilator responses to the two activators A769662 and acetylcholine as those from normoxic mice, although the contribution of an increase in nitric oxide production to uterine artery vasodilation was reduced in the hypoxic vs normoxic groups. Vasoconstrictor responses to phenylephrine and potassium chloride were unaltered by hypoxia. The levels of activated adenosine monophosphate-activated protein kinase (AMPK) were reduced with hypoxia in both the uterine artery and placenta as measured by western blot and immunohistochemistry. We concluded that the rise in uterine artery blood flow may be compensatory to hypoxia but was not sufficient to prevent fetal growth restriction. Although AMPK signaling was reduced by hypoxia, AMPK was still receptive to pharmacologic activation in the uterine arteries in which it was a potent vasodilator. Thus, AMPK activation may represent a new therapy for pregnancy complications involving reduced uteroplacental perfusion.

Pharmacological activation of peroxisome proliferator-activated receptor γ (PPAR-γ) protects against hypoxia-associated fetal growth restriction.

The hypoxia of high-altitude (HA) residence increases the risk of intrauterine growth restriction (IUGR) and preeclampsia 3-fold, augmenting perinatal morbidity and mortality and the risk for childhood and adult disease. Currently, no effective therapies exist to prevent these vascular disorders of pregnancy. The peroxisome proliferator-activated receptor γ (PPAR-γ) is an important regulator of uteroplacental vascular development and function and has been implicated in the pathogenesis of IUGR and preeclampsia. Here, we used a model of HA pregnancy in mice to determine whether hypoxia-induced fetal growth restriction reduces placental PPAR-γ protein expression and placental vascularization and, if so, to evaluate the effectiveness of the selective PPAR-γ agonist pioglitazone (PIO) for preventing hypoxia-induced IUGR. Hypoxia resulted in asymmetric IUGR, placental insufficiency, and reduced placental PPAR-γ expression; PIO prevented approximately half of the fetal growth restriction and attenuated placental insufficiency. PIO did not affect fetal growth under normoxia. Although PIO was beneficial for fetal growth, PIO treatment reduced placental vascular density of the labrynthine zone in normoxic and hypoxic (Hx) conditions, and mean vascular area was reduced in the Hx group. Our results suggest that pharmacological PPAR-γ activation is a potential strategy for preventing IUGR in pregnancies complicated by hypoxia, although further studies are needed to identify its likely metabolic or vascular mechanisms.-Lane, S. L., Dodson, R. B., Doyle, A. S., Park, H., Rathi, H., Matarrazo, C. J., Moore, L. G., Lorca, R. A., Wolfson, G. H., Julian, C. G. Pharmacological activation of peroxisome proliferator-activated receptor γ (PPAR-γ) protects against hypoxia-associated fetal growth restriction.

High Altitude Reduces NO-Dependent Myometrial Artery Vasodilator Response During Pregnancy.

The chronic hypoxia of high-altitude (HA) residence reduces uterine artery blood flow during pregnancy, likely contributing to an increased frequency of preeclampsia and intrauterine growth restriction. We hypothesized that this lesser pregnancy blood flow rise was due, in part, to reduced vasodilation of myometrial arteries (MAs). Here, we assessed MA vasoreactivity in healthy residents of high (2902±39 m) or low altitude (LA; 1669±10 m). MA contractile responses to potassium chloride, phenylephrine, or the thromboxane A2 agonist U46619 did not differ between LA and HA women. Acetylcholine vasodilated phenylephrine or U466119 preconstricted MAs at LA, yet had no effect on HA MAs. In contrast, another vasodilator, bradykinin, relaxed MAs from both altitudes similarly. At LA, the NO synthase inhibitor L-NG-nitroarginine methyl ester decreased both acetylcholine and bradykinin vasodilation by 56% and 33%, respectively. L-NG-nitroarginine methyl ester plus the COX (cyclooxygenase) inhibitor indomethacin had similar effects on acetylcholine and bradykinin vasodilation (68% and 42% reduction, respectively) as did removing the endothelium (78% and 50% decrease, respectively), suggesting a predominantly NO-dependent vasodilation at LA. However, at HA, L-NG-nitroarginine methyl ester did not change bradykinin vasodilation, whereas indomethacin or endothelium removal decreased it by 28% and 72%, respectively, indicating impaired NO signaling at HA. Suggesting that the impairment was downstream of eNOS (endothelial NO synthase), HA attenuated the vasodilation elicited by the NO donor sodium nitroprusside. We concluded that reduced NO-dependent MA vasodilation likely contributes to diminished uteroplacental perfusion in HA pregnancies.