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Purpose: This study aims to assess the diagnostic utility and provide reporting recommendations for clinical DNA methylation episignature testing based on the cohort of patients tested through the EpiSign Clinical Testing Network., Methods: The EpiSign assay utilized unsupervised clustering techniques and a support vector machine-based classification algorithm to compare each patient's genome-wide DNA methylation profile with the EpiSign Knowledge Database, yielding the result that was reported. An international working group, representing distinct EpiSign Clinical Testing Network health jurisdictions, collaborated to establish recommendations for interpretation and reporting of episignature testing., Results: Among 2399 cases analyzed, 1667 cases underwent a comprehensive screen of validated episignatures, imprinting, and promoter regions, resulting in 18.7% (312/1667) positive reports. The remaining 732 referrals underwent targeted episignature analysis for assessment of sequence or copy-number variants (CNVs) of uncertain significance or for assessment of clinical diagnoses without confirmed molecular findings, and 32.4% (237/732) were positive. Cases with detailed clinical information were highlighted to describe various utility scenarios for episignature testing., Conclusion: Clinical DNA methylation testing including episignatures, imprinting, and promoter analysis provided by an integrated network of clinical laboratories enables test standardization and demonstrates significant diagnostic yield and clinical utility beyond DNA sequence analysis in rare diseases., Competing Interests: Conflict of Interest Bekim Sadikovic is a shareholder in EpiSign Inc, company involved in commercialization of EpiSign technology. All other authors declare no conflicts of interest., (Copyright © 2024 American College of Medical Genetics and Genomics. Published by Elsevier Inc. All rights reserved.)

The present study was designed to (1) assess the role of triiodothyronine (T(3)) with regard to in vitro steroid hormone secretion by chicken ovarian follicles; (2) determine whether T(3) influences the in vivo function of the pituitary-ovarian axis in the hen; and (3) detect expression of thyroid hormone receptor (TR) mRNA in chicken ovarian follicles. In the first experiment, laying hens were decapitated 22.5h before ovulation. White prehierarchical follicles (1-8mm) and fragments of theca and granulosa layers of the 3 largest yellow preovulatory follicles F3-F1 (22-35mm) were incubated in a medium supplemented with T(3) (0, 0.1, 1, 10, 100, or 1000ng/mL) or ovine luteinizing hormone (LH) (10ng/mL) in combination with doses of T(3) (1, 10, and 100ng/mL). Triiodothyronine decreased basal and LH-stimulated estradiol secretion by white follicles and the theca layer of all preovulatory follicles. On the other hand, it increased progesterone secretion by F2 and F1 follicles. In the second experiment, hens were injected 1h after ovulation with saline (control) or T(3) (10microg/100g body weight, intraperitoneally). Results indicated that exogenous T(3) decreased plasma concentrations of LH and estradiol and increased plasma concentrations of progesterone. In the third experiment, using reverse transcription polymerase chain reaction (RT-PCR) analysis, expression of thyroid hormone receptor (TRalpha and TRbeta0), mRNA was detected in all of the ovarian compartments. The expression of TRalpha mRNA was relatively greater in comparison with TRbeta0. There were no differences between white ovarian follicles in the expression of TRalpha and TRbeta0 mRNA. A considerably higher TRalpha and lower TRbeta0 expression was detected in the granulosa layer of preovulatory follicles in comparison with the theca layer. In conclusion, the data indicate that thyroid hormones acting via nuclear receptors are involved in regulation of the pituitary-ovarian axis and processes associated with follicle growth and maturation.

The present study was undertaken to examine the effect of recombinant chicken leptin administered to fed ad libitum and feed-restricted immature chickens of a layer strain on ovarian development and the timing of sexual maturity. In the first experiment 11-week-old pullets (77 days of age) fed ad libitum were injected daily with leptin at four dose levels (4, 16, 64 and 256 microg/kg body weight) until sexual maturity (lay of the first egg). Leptin treatment at the highest dose significantly (P<0.05) advanced the onset of puberty (day 116.3+/-1.0) in comparison to controls (day 121.3+/-1.2). The rises of luteinizing hormone, estradiol and progesterone in blood plasma were also advanced by leptin treatment. In the second experiment, both full-fed and feed-restricted pullets (79 days of age) were injected daily with leptin (256 microg/kg body weight). In birds fed ad libitum, exogenous leptin again significantly (P<0.05) advanced first ovipostion (day 118.4+/-1.4 versus day 124.4+/-1.7), while abolishing the significant (P<0.05) delay caused by feed restriction (day 131.5+/-1.6) and restoring the normal onset of sexual maturity (day 125.7+/-1.6). Analysis of the ovaries in 106-day-old pullets revealed that leptin injections advanced follicular development, particularly in birds fed ad libitum, and significantly (P<0.01) reduced follicular apoptosis both in full-fed and feed-restricted birds. In conclusion, we have shown that in female chickens exogenous leptin advances the onset of puberty by attenuation of ovarian apoptosis and enhancement of folliculogenesis.

The effect of growth hormone (GH) on insulin-like growth factor I (IGF-I) secretion and the effects of GH and IGF-I on [(3)H] thymidine incorporation and estradiol (E2) secretion by theca interna (Tc) and granulosa cells (Gc) cultured alone and in coculture were studied in cultured porcine follicular cells, prepared from small (SF), medium (MF) and large (LF) preovulatory follicles. We demonstrated that both Tc and Gc secrete IGF-I and that GH had no effect on IGF-I secretion by Tc but, increased IGF-I secretion by Gc isolated from SF and cultured alone or in coculture. IGF-I stimulated secretion of E2 by all cells, except in Tc derived from SF in which the effect was not statistically significant. The only stimulatory effect of concurrent treatment with GH on E2 secretion was noted in Tc derived from MF. IGF-I increased the [(3)H] thymidine incorporation in all Tc cells but GH did not augment this effect. In Gc, IGF-I stimulated [(3)H] thymidine incorporation in cells derived from SF and MF but not from LF. GH had no stimulatory effect except on Gc derived from LF and grown alone. The highest stimulatory effect was observed in SF. This was smaller in MF and no effect was noted in LF. In conclusion, our work shows that both Gc and Tc are sites of IGF-I production and for the first time shows the stimulatory role of IGF-I in proliferation of Tc cells derived from all types of follicles and augmentation of E2 secretion in Tc derived from MF and LF. The promotion of the mitogenic activity in Tc by IGF-I during all stages of follicular development suggests an important role for theca cells in follicular growth.

The purpose of the experiment was to obtain preparatory information about the presence of prostaglandins in semen collected from various types of horses after different periods of sexual rest. Semen was collected with an artificial vagina. Prostaglandin-like activity was estimated by the bioassay procedure described by Vane (1). Results are expressed in ng/ml PGE(2) of seminal plasma. The total concentration of prostaglandins in the full ejaculate averaged 43.73 +/- 4.93 ng/ml of plasma while the total amount of prostaglandins in the ejaculate was 1076 ng. Taking into consideration the period of sexual rest in the stallion, statistically significant differences were found in the prostaglandin level in the semen of all the stallions.