Primordial follicle activation is a process in which individual primordial follicles leave their dormant state and enter a growth phase. While existing hormone stimulation strategies targeted the growing follicles, the remaining dormant primordial follicles were ruled out from clinical use. Recently, in vitro activation (IVA), which is a method for controlling primordial follicle activation, has provided an innovative technology for primary ovarian insufficiency (POI) patients. IVA was developed based on Hippo signaling and phosphatase and tensin homolog (PTEN)/phosphatidylinositol- 3-kinase (PI3K)/protein kinase B (AKT)/forkhead box O3 (FOXO3) signaling modulation. With this method, dormant primordial follicles are activated to enter growth phase and developed into competent oocytes. IVA has been successfully applied in POI patients who only have a few remaining remnant primordial follicles in the ovary, and healthy pregnancies and deliveries have been reported. IVA may also provide a promising option for fertility preservation in cancer patients and prepubertal girls whose fertility preservation choices are limited to tissue cryopreservation. Here, we review the basic mechanisms, translational studies, and current clinical results for IVA. Limitations and further study requirements that could potentially optimize IVA for future use will also be discussed.

Kim MK, et al. J Korean Med Sci. 2019 Jan;34(4):e27. https://doi.org/10.3346/jkms.2019.34.e27

The uterus is dynamically regulated in response to various signaling triggered by hormones during the estrous cycle. The Hippo signaling pathway is known as an important signaling for regulating cellular processes during development by balancing between cell growth and apoptosis. Serine/threonine protein kinase 3/4 (STK3/4) is a key component of the Hippo signaling network. However, the regulation of STK3/4-Hippo signaling in the uterus is little known. In this study, we investigated the regulation and expression of STK3/4 in the uterine endometrium during the estrous cycle. STK3/4 expression was dynamically regulated in the uterus during the estrous cycle. STK3/4 protein expression was gradually increased from the diestrus stage and reached the highest in the estrus stage. STK3/4 was exclusively localized in the luminal and glandular epithelial cells of the uterus, and phosphorylated STK3/4 was also increased at the estrus stage. Moreover, the increase of STK3/4 expression in uteri was induced by administration of estradiol, but not by progesterone injection in ovariectomized mice. Pretreatment with an estrogen receptor antagonist ICI 182,780 reduced estrogen-induced STK3/4 expression and its phosphorylation. The estrogen-induced STK3/4 expression was related to the increase in phosphorylation of downstream targets including LATS1/2 and YAP. These findings suggest that STK3/4-Hippo signaling acts a novel signaling pathway in the uterine epithelium and STK3/4-Hippo is one of key molecules for connecting between the estrogen downstream signaling pathway and the Hippo signaling pathway leading to regulate dynamic uterine epithelium during the estrous cycle.

Peroxisome proliferator-activated receptor gamma (PPARγ) is known as a regulator of cellular functions, including adipogenesis and immune cell activation. The objectives of this study were to investigate the expression of PPARγ and identify the mechanism of primordial follicle activation via PPARγ modulators in mouse ovaries. We first measured the gene expression of PPARγ and determined its relationship with phosphatase and tensin homolog (PTEN), protein kinase B (AKT1), and forkhead box O3a (FOXO3a) expression in neonatal mouse ovaries. We then incubated neonatal mouse ovaries with PPARγ modulators, including rosiglitazone (a synthetic agonist of PPARγ), GW9662 (a synthetic antagonist of PPARγ), and cyclic phosphatidic acid (cPA, a physiological inhibitor of PPARγ), followed by transplantation into adult ovariectomized mice. After the maturation of the transplanted ovaries, primordial follicle growth activation, follicle growth, and embryonic development were evaluated. Finally, the delivery of live pups after embryo transfer into recipient mice was assessed. While PPARγ was expressed in ovaries from mice of all ages, its levels were significantly increased in ovaries from 20-day-old mice. In GW9662-treated ovaries in vitro, PTEN levels were decreased, AKT was activated, and FOXO3a was excluded from the nuclei of primordial follicles. After 1 month, cPA-pretreated, transplanted ovaries produced the highest numbers of oocytes and polar bodies, exhibited the most advanced embryonic development, and had the greatest blastocyst formation rate compared to the rosiglitazone- and GW9662-pretreated groups. Additionally, the successful delivery of live pups after embryo transfer into the recipient mice transplanted with cPA-pretreated ovaries was confirmed. Our study demonstrates that PPARγ participates in primordial follicle activation and development, possibly mediated in part by the PI3K/AKT signaling pathway. Although more studies are required, adapting these findings for the activation of human primordial follicles may lead to treatments for infertility that originates from poor ovarian reserves.

Background/Aims: Cyclic adenosine monophosphate (cAMP)-dependent type 2 regulatory subunit beta (Prkar2b) is a regulatory isoform of cAMP-dependent protein kinase (PKA), which is the primary target for cAMP actions. In oocytes, PKA and the pentose phosphate pathway (PPP) have important roles during the germinal vesicle (GV) stage arrest of development. Although the roles of the PKA signal pathway have been studied in the development of oocyte, there has been no report on the function of PRKAR2B, a key regulator of PKA. Methods: Using reverse transcription polymerase chain reaction (RT-PCR), quantitative real-time PCR (qRT-PCR), immunohistochemistry, and immunofluorescence, we determined the relative expression of Prkar2b in various tissues, including ovarian follicles, during oocyte maturation. Prkar2b-interfering RNA (RNAi) microinjection was conducted to confirm the effect of Prkar2b knockdown, and immunofluorescence, qRT-PCR, and time-lapse video microscopy were used to analyze Prkar2b-deficient oocytes. Results: Prkar2b is strongly expressed in the ovarian tissues, particularly in the growing follicle. During oocyte maturation, the highest expression of Prkar2b was during metaphase I (MI), with a significant decrease at metaphase II (MII). RNAi-mediated Prkar2b suppression resulted in MI-stage arrest during oocyte development, and these oocytes exhibited abnormal spindle formation and chromosome aggregation. Expression of other members of the PKA family (except for Prkaca) were decreased, and the majority of the PPP factors were also reduced in Prkar2b-deficient oocytes. Conclusion: These results suggest that Prkar2b is closely involved in the maturation of oocytes by controlling spindle formation and PPP-mediated metabolism.

As paternal age increases, the quality of sperm decreases due to increased DNA fragmentation and aneuploidy. Higher levels of structural chromosomal aberrations in the gametes ultimately decrease both the morphologic quality of embryos and the pregnancy rate. In this study, we investigated whether paternal age affected the euploidy rate. This study was performed using the medical records of patients who underwent in vitro fertilization (IVF) procedures with preimplantation genetic screening (PGS) from January 2016 to August 2017 at a single center. Based on their morphological grade, embryos were categorized as good- or poor-quality blastocysts. The effects of paternal age were elucidated by adjusting for maternal age. Among the 571 total blastocysts, 219 euploid blastocysts were analyzed by PGS (38.4%). When the study population was divided into four groups according to both maternal and paternal age, significant differences were only noted between groups that differed by maternal age (group 1 vs. 3, p=0.031; group 2 vs. 4, p=0.027). Further analysis revealed no significant differences in the euploidy rate among the groups according to the morphological grade of the embryos. Paternal age did not have a significant impact on euploidy rates when PGS was performed. An additional study with a larger sample size is needed to clarify the effects of advanced paternal age on IVF outcomes.