In Vitro Grown Sheep Preantral Follicles Yield Oocytes with Normal Nuclear-Epigenetic Maturation

BACKGROUND: Assisted reproductive technologies allow to utilize a limited number of fully grown oocytes despite the presence in the ovary of a large pool of meiotically incompetent gametes potentially able to produce live births. In vitro folliculogenesis could be useful to recruit these oocytes by promoting their growth and differentiation. METHODOLOGY/PRINCIPAL FINDINGS: In vitro folliculogenesis was performed starting from sheep preantral (PA) follicles to evaluate oocyte nuclear/epigenetic maturation. Chromatin configuration, quantification of global DNA methylation, and epigenetic remodelling enzymes were evaluated with immunocytochemistry, telomere elongation was assessed with the Q-FISH technique, while the DNA methylation status at the DMRs of maternally IGF2R and BEGAIN, and paternally H19 methylated imprinted genes was determined by bisulfite sequencing and COBRA. Specifically, 70% of PA underwent early antrum (EA) differentiation and supported in culture oocyte global DNA methylation, telomere elongation, TERT and Dnmt3a redistribution thus mimicking the physiological events that involve the oocyte during the transition from secondary to tertiary follicle. Dnmt1 anticipated cytoplasmic translocation in in vitro grown oocytes did not impair global and single gene DNA methylation. Indeed, the in vitro grown oocytes acquired a methylation profile of IGF2R and BEGAIN compatible with the follicle/oocyte stage reached, and maintained an unmethylated status of H19. In addition, the percentage of oocytes displaying a condensed chromatin configuration resulted lower in in vitro grown oocytes, however, their ability to undergo meiosis and early embryo development after IVF and parthenogenetic activation was similar to that recorded in EA follicle in vivo grown oocytes. CONCLUSIONS/SIGNIFICANCE: In conclusion, the in vitro folliculogenesis was able to support the intracellular/nuclear mechanisms leading the oocytes to acquire a meiotic and developmental competence. Thus, the in vitro culture may increase the availability of fertilizable oocytes in sheep, and become an in vitro translational model to investigate the mechanisms governing nuclear/epigenetic oocyte maturation

Impact of COVID-19 on cardiovascular testing in the United States versus the rest of the world

Objectives: This study sought to quantify and compare the decline in volumes of cardiovascular procedures between the United States and non-US institutions during the early phase of the coronavirus disease-2019 (COVID-19) pandemic. Background: The COVID-19 pandemic has disrupted the care of many non-COVID-19 illnesses. Reductions in diagnostic cardiovascular testing around the world have led to concerns over the implications of reduced testing for cardiovascular disease (CVD) morbidity and mortality. Methods: Data were submitted to the INCAPS-COVID (International Atomic Energy Agency Non-Invasive Cardiology Protocols Study of COVID-19), a multinational registry comprising 909 institutions in 108 countries (including 155 facilities in 40 U.S. states), assessing the impact of the COVID-19 pandemic on volumes of diagnostic cardiovascular procedures. Data were obtained for April 2020 and compared with volumes of baseline procedures from March 2019. We compared laboratory characteristics, practices, and procedure volumes between U.S. and non-U.S. facilities and between U.S. geographic regions and identified factors associated with volume reduction in the United States. Results: Reductions in the volumes of procedures in the United States were similar to those in non-U.S. facilities (68% vs. 63%, respectively; p = 0.237), although U.S. facilities reported greater reductions in invasive coronary angiography (69% vs. 53%, respectively; p < 0.001). Significantly more U.S. facilities reported increased use of telehealth and patient screening measures than non-U.S. facilities, such as temperature checks, symptom screenings, and COVID-19 testing. Reductions in volumes of procedures differed between U.S. regions, with larger declines observed in the Northeast (76%) and Midwest (74%) than in the South (62%) and West (44%). Prevalence of COVID-19, staff redeployments, outpatient centers, and urban centers were associated with greater reductions in volume in U.S. facilities in a multivariable analysis. Conclusions: We observed marked reductions in U.S. cardiovascular testing in the early phase of the pandemic and significant variability between U.S. regions. The association between reductions of volumes and COVID-19 prevalence in the United States highlighted the need for proactive efforts to maintain access to cardiovascular testing in areas most affected by outbreaks of COVID-19 infection

Incidence of the different patterns of chromatin configuration in <i>in vitro</i> and <i>in vivo</i> grown oocytes.

<p>The percentages of chromatin configurations in oocytes isolated from PA, <i>in vivo</i> and <i>in vitro</i> grown EA and preovulatory follicles. Different superscripts denote values with highly significative (<i>P</i><0.01) differences (χ2 test).</p><p>Data are expressed as percentage.</p><p>a,b,c,d = different superscripts denote differences among groups.</p><p>A,B,C = different superscripts denote differences within groups (NSN vs. SN vs. SNE).</p

Chromatin remodelling and global DNA methylation in <i>in vitro</i> and <i>in vivo</i> grown oocytes.

<p>Digital images of sheep germinal vesicles (GVs) showing different chromatin configurations (left images), global DNA methylation patterns (middle images), and merged images (right images). Chromatin counterstaining was performed with SYBR Green 14/I (green), which detects double-stranded DNA, while global DNA methylation was analyzed with a 5-methylcytosine antibody (red). (<b>A</b>) A typical example of a GV germ cell obtained from a preantral (PA) follicle with a diffuse chromatin not surrounding the nucleolus (not surrounded nucleolus configuration: NSN) that did not display any immunostaining for 5-methylcytosine (A1,A2). (<b>B,C</b>) oocytes obtained from <i>in vitro</i> grown early antral (EA) follicles with either a NSN (B) or a surrounded nucleolus (SN) chromatin configuration (C), respectively. As observed in oocytes obtained from <i>in vivo</i> grown EA follicles, in both categories of <i>in vitro</i> grown oocytes the 5-methylcytosine immunopositivity indicates a comparable degree of global DNA methylation (NSN = B1,B2; SN = C1,C2). (<b>D</b>) GV oocyte isolated from a preovulatory follicle with a condensed chromatin localized surrounding the nucleolus and the nuclear envelope (SNE configuration) with a clear merged positivity with 5-methylcytosine antibody (D1,D2). Scale bar = 10 µm.</p

Overall methylation status of the <i>IGF2R</i> and <i>BEGAIN</i> genes in germ cells analyzed by COBRA.

<p>The same DNA amplified by PCR and used for bisulfite sequencing was digested by enzymes that cut only if the site was methylated. The size of the expected fragments for the unmethylated (<) and methylated (◂) alleles are indicated. The undigested and digested products are indicative of unmethylated and methylated templates respectively. Combined bisulfite restriction analysis (COBRA) showed on overall methylation pattern comparable to the one obtained by bisulfite sequencing. In those samples in which there are both methylated and unmethylated clones (e.g. GV and PA for <i>IGF2R</i> and MII oocytes for <i>BEGAIN</i>) the specific restriction enzyme cutting sites are preferentially methylated, resulting only in cleaved products after enzymatic digestion. m = 100 bp Marker; uncut = undigest PCR product. Cleavage CpG sites (CpGs) are indicated: <i>BstUI</i> (CG/CG) cuts between CpGs 3 and 4, 10 and 11 in <i>IGF2R</i>. <i>HhaI</i> (C/GCG) cuts at CpG 6 in <i>BEGAIN</i>. Preov, preovulatory follicles isolated from hormonal treated sheep.</p

Ooocyte nuclear stage recorded in different categories of oocytes after IVM.

<p>Values are the means ± s.d. Different letters within each column indicate statistically significant differences (<i>P</i><0.01, ANOVA test). n°, total number of oocytes analyzed; the number of degenerated oocytes was subtracted from the total number. GV, germinal vesicle; GVBD/MI, germinal vesicle breakdown/metaphase I; MII, metaphase II; PA, preantral follicles; EA, early antral follicles; A, antral follicles.</p

Parthenogenetic activation of MII oocytes obtained from different categories of follicles.

<p>Values are the means ± s.d. Different letters within each column indicate statistically significant differences (<i>P</i><0.01, ANOVA test). n°, total number of oocytes analyzed. The degenerated oocytes were subtracted from the total number of oocytes. EA, early antral follicles; A, antral follicles.</p

Dnmt3a nuclear distribution pattern during <i>in vitro</i> and <i>in vivo</i> oocyte growth.

<p>Examples of digital images showing Dnmt3a distribution pattern visualized with a Cy3 secondary antibody (red), while DNA was counterstained with SYBR Green 14/I (green). Dnmt3a maintained a nuclear localization in all <i>in vitro</i> and <i>in vivo</i> classes of germ cells analyzed. (<b>A,B</b>) Oocyte obtained from a preantral (PA) follicle (A) and from an <i>in vitro</i> grown oocyte isolated from an early antral (EA) follicle (B) with a not surrounded nucleolus (NSN) configuration: Dnmt3a is homogeneously distributed within the nucleus with a clustered arrangement in the vicinity of the DNA. (<b>C</b>) <i>In vitro</i> grown oocyte collected from an EA follicle which displays a surrounded nucleolus (SN) chromatin configuration: Dnmt3a is associated with DNA or gathered in clusters around it. (<b>D</b>) Oocyte isolated from a preovulatory follicle where Dnmt3a is strictly associated with the condensed chromatin. Scale bar = 5 µm.</p

Methylation status of the <i>IGF2R</i> and <i>BEGAIN</i> genes in germ cells analyzed by bisulfite sequencing.

<p>An example of bisulphite sequencing analysis of examined CpGs in three imprinted genes. Each line represents an individual sequence molecule, with each circle corresponding to a separate CpG site (CpG). Black and white circles indicate methylated and unmethylated CpGs respectively. Arrows indicate the CpGs assayed by <i>BstUI</i> and <i>HhaI</i> enzymes, used in the combined bisulfite restriction analysis (COBRA). GV, germinal vesicle; MII, metaphase II; IVM, <i>in vitro</i> maturation; PA, preantral follicles; EA, early antral follicles; Preov, preovulatory follicles; A, antral follicles. Percentages of methylation were calculated by counting the number of methylated CpGs out of the total number of CpGs within the fragment amplified.</p