• Clinical and Experimental Reproductive Medicine
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• v.46 no.2
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• pp.43-49
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• 2019

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.

• Journal of Animal Reproduction and Biotechnology
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• v.36 no.1
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• pp.51-58
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• 2021

Kisspeptin, a neuropeptide and the master controller of reproductive axis upstream to GnRH neurons, and its receptor are also expressed in extra-hypothalamic tissues, such as ovaries. As systemic kisspeptin has been shown to modulate follicular dynamics in cattle, we hypothesized that kisspeptin has direct actions on the ovarian follicular development. We also hypothesized that kisspeptin regulation of primordial follicle development is via modulation of VEGF expression. In order to test these hypotheses, we cultured caprine ovarian cortical strips in vitro for 7 days with supplementation of kisspeptin at 1, 10 and 100 µM concentration and observed the development of primordial follicles into intermediate, primary and secondary follicles. We also studied the alteration in the expression profile of VEGF and VEGF transcript variant 2 mRNA during follicular development in the presence of kisspeptin. We confirmed the presence of GPR54 in goat ovaries in our preliminary studies. Supplementation of kisspeptin at 1 and 10 µM concentration facilitated the development of primordial follicles into intermediate, primary and secondary follicles with less number of degenerated follicles while the same at 100 µM resulted in degeneration of follicles. We observed a drastic increase in the expression profile of VEGF and VEGF transcript variant 2 mRNA upon culture which was independent of kisspeptin treatment. In conclusion, our studies show that kisspeptin facilitates ovarian primordial development in vitro.

• Journal of Animal Reproduction and Biotechnology
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• v.34 no.1
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• pp.40-49
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• 2019

Chronic and unpredictable stress can disrupt the female reproductive system by suppression for secretion of gonadotrophin-releasing hormone (GnRH) and gonadotrophin, resulted in ovarian malfunction and infertility. In the recent days, kisspeptin has been highly highlighted as a hypothalamic peptide which directly stimulates synthesis and release for GnRH. However, in spite of the key role of kisspeptin in the female reproductive system, little information is still available on the changes of its expression during ovarian cycle under stressed condition. Therefore, we induced chronic and unpredictable stress series to the female mice to analyze kisspeptin expression in the brain and ovary. Stressed mice exhibited changes of behavior and body weight gain during the stress assessment, which suggested that the present stress model in mice was successfully established. In the brain level, kisspeptin expression was attenuated than control. In the ovary level, the stressed mice displayed irregularly shrunk oocytes with broken zona pellucida throughout the follicle stages, pyknotic granulosa cells, decreased number of developing follicles and increased number of atretic follicles than the control. In case of kisspeptin expression in the whole ovary tissue, the expression level was decreased in the stressed mice. In detail, the less intensity of kisspeptin expression in the antral follicles phase was observed in the stressed mice than control mice, indicating that local function of kisspeptin during ovary cycle is highly associated with development of ovarian follicles. We expect that the present study has important implications for the fields of reproductive biology.

• Asian-Australasian Journal of Animal Sciences
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• v.19 no.12
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• pp.1711-1715
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• 2006

To predict the number of preantral (primordial, primary and secondary) follicles retrieved from bovine ovaries, we examined the relationship between morphological parameters of ovaries and number of preantral follicles retrieved mechanically. The preantral follicles were retrieved mechanically by slicing ovarian tissue and the influences of size of the ovaries, number of antral follicles, and presence of cystic follicle and corpus luteum on the retrieval were evaluated. Total 77 ovaries were used and significant (p<0.05) relationship was detected between the number of antral follicles and the presence of cystic follicles, and the retrieval number. More preantral follicles were retrieved from the ovaries having more than 20 antral follicles than those having less than 20 antral follicles (17,760${\pm}$5,637 vs. 3,689${\pm}$537) in the ovarian cortex. The retrieval number was significantly reduced in cystic ovaries compared with non-cystic ovaries (5,167${\pm}$825 vs. 20,631${\pm}$6,507). However, neither ovary size (<3.5, 3.5 to 4.0, 4.0 to 4.5 and >4.5 cm) nor the presence of corpus luteum affected the follicle retrieval. In conclusion, the number of preantral follicles retrieved from the ovaries can simply be predicted by the number of antral follicles and the presence of cystic follicles in the ovarian cortex.

• Clinical and Experimental Reproductive Medicine
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• v.26 no.2
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• pp.251-256
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• 1999

The present study was conducted to evaluate whether vitrification could be used for ovarian tissue preservation. The important issue here is that the vitrification is very simple, easy, and economical compared to the conventional cryopreserving method that using automatic freezing instrument. Human ovarian cortical tissues were cryopreserved by vitrification with 5.5 M ethylene glycol and 1.0 M sucrose as cryoprotectant. Three points of temperature ($4^{\circ}C$, room temperature, and $37^{\circ}C$) and two points of duration (5 or 10 minutes) for cryoprotectant treatment were examined to determine the best condition for vitrification of the human ovarian cortical tissues. After thawing, viability of the isolated primordial follicles was examined by dye-exclusion method. Histological appearance of tissues before and after the cryopreservation was evaluated. There was no toxic effect of the 5.5 M ethylene glycol on the primordial follicles. However, when the tissues were treated with cryoprotectant at $37^{\circ}C$ for 10 minutes and exposed to liquid nitrogen, it seems likely that there is certain deleterious effects on the viability of the primordial follicles. The highest viability of the primordial follicles was obtained with the treatment of cryoprotectant at room temperature for 10 minutes. Follicles and oocytes survived after freezing and thawing had the similar normal shapes as was seen in the specimens before cryopreservation. It would be useful to apply vitrification in establishing ovarian tissue banking for clinical purposes.

• Clinical and Experimental Reproductive Medicine
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• v.16 no.1
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• pp.81-91
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• 1989

It has been suggested that the prognosis for fertility of the infertile patients with healed pelvic tuberculosis is very poor. Total 60 patients(77 cycles) with previous history of pelvic tuberculosis who underwent IVF-ET from January 1988 to March 1989 at SNUH were classified into three groups according to the principal histopathological lesions : tuberculous endometritis group(N=20, 28 cycles), tuberculous salpingitis group(N=32, 37 cycles) and pelvic peritoneal tuberculosis group(N=8, 12 cycles). To evaluate the effects of previous pelvic tuberculous lesions on ovarian follicular growth and development in controlled ovarian hyperstimulation for IVF-ET and its final outcome, serum E2 levels on the day of hCG administration(Day 0) and the day after hCG administration(Day +1), the number of ovarian follicles with mean diamete ${\geqq}$ 12 mm on Day 0, the number of oocytes retrieved by transvaginal aspiration, and pregnancy rate per cycle were measured and compared with control group(N=123, 161 cycles). There were no significant differences in cancellation rate during controlled ovarian hyperstimulation, total dosage of FSH and hMG administrated, menstrual cycle date(MCD) of hCG injection, serum E2 levels, the number of ovarian follicles with mean diameter ${\geqq}$ 15 mm, and the number of oocytes retrieved between pelvic tuberculosis group and control group. But in pelvic tuberculosis group, the number of ovarian follicles with mean diameter 12-14 mm, total number of ovarian follicles(${\geqq}$ 12 mm), and pregnancy rate per cycle were significantly decreased. These data suggest that previous pelvic tuberculous lesions have no significant adverse effects on the ovarian response to gonadotropin stimulation. IVF-ET proved to be an useful treatment modality for infertile patients with previous history of pelvic tuberculosis in spite of its relatively lowered pregnancy rate.

• Clinical and Experimental Reproductive Medicine
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• v.27 no.2
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• pp.145-149
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• 2000

Objective: The present study was conducted to evaluate the viability of germ cells from the adult and fetal ovarian tissues after vitrification followed by xenografting. Method: The human adult ovarian tissues were obtained from 33 years old patient, and the fetal ovarian tissues were obtained from 22 weeks and 25 weeks in gestation. Ovarian tissues were cryopreserved by vitrification with 5.5 M ethylene glycol (EG 5.5) and 1.0 M sucrose as cryoprotectants. Adult and fetal ovarian tissues were pre-equilibrated with EG 5.5 at room temperature for 10 and 5 minutes, respectively and plunged into liquid nitrogen immediately. Frozen-thawed tissues were xenografted into NOD-SCID mice to evaluate the viability and capacity for further growth of the primordial follicles. Grafts were recovered from the recipients 4 weeks after transplantation and histological analysis was accomplished. Result and Conclusion: Grafts recovered 4 weeks after transplantation contained less number of oocytes and primordial follicles compared to that of the fresh tissues. Survived follicles were mainly primordial and intermediary with larger diameter and more granulosa cells. It is confirmed that 1) the ovarian tissues were healthy and the germ cells were survived after vitrification, and 2) the survived fetal primordial follicles after vitrification resumed the growth in the xenografts.

• Clinical and Experimental Reproductive Medicine
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• v.29 no.3
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• pp.195-200
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• 2002

Objective : To investigate the expression of apoptosis related proteins and apoptotic cells on the human ovarian follicles. Materials and Methods: Thirty five Formalin-fixed paraffin-embedded human ovarian tissue blocks were selected from the surgical pathology files of the department of pathology, College of Medicine, Yonsei University, for the period from 1996 to 1998. All specimen were from premenopausal women aged from $32{\sim}45$. Ovarian tissues were collected from the patients performing hysterectomy for benign uterine diseases. Immunohistochemical staining was performed for the detection of DNA fragmented cell, Bcl-2, Bax, Fas and Fas-ligand. Results: Bcl-2 and bax were not expressed on the surrounding cells and oocyte of the primary, primordial and preantral follicles. Fas and Fas-ligand (Fas-L) were not expressed on the surrounding cells on the primordial and primary follicles. But expressed on the surrounding granulosa cells and oocyte in the primordial and primary follicles. In the healthy follicles, Bcl-2 was expressed on the granulosa cells, however, Bax was not expressed. DNA fragmented cells were expressed on the inner granulosa cell layer of atretic follicles. Conclusion: Fas, Fas-ligand, and Bax may be responsible for the follicular atresia and Bcl-2 may be involved in the follicular survival in the human ovary.

• Journal of Veterinary Clinics
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• v.5 no.2
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• pp.83-86
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• 1988

Morphometric observation on the ovarian follicles of ICR strain-female mice has been conducted to investigate the effect of induced inflammation on the follicular populations. Inflammation was induced by administering 25${\mu}\ell$ of turpentine oil intraperitoneally 2 times at the interval of 3 days. Mice showing 5-day estrous cycle about 20 days after induction of inflammation are placed in experimental group. Normal follicles of 100～399$\mu\textrm{m}$ in diameter increased at estrus but those of over 400$\mu\textrm{m}$ decreased. super-ovulation increased the number of normal follicles of over 400$\mu\textrm{m}$. The number of normal follicles of over 400$\mu\textrm{m}$ 48 hours after superovulation was not significantly different between experimental and control groups. Present results indicated that the number of preovulatory follicles 48 hours after PMSG injection did not decrease in the mice which showed regular estrous cycle even after the induction of inflammation.

• Clinical and Experimental Reproductive Medicine
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• v.15 no.2
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• pp.119-128
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• 1988

It has been suggested that the presence of periovarian adhesions might impair the ovarian response to gonadotropins. Total 136 patients who underwent IVF-ET from February to June 1988(88-1 and 88-2 series) at SNUH were classified into three groups according to total ovarian access score, sum of each ovarian availability, estimated by diagnostic laparoscopy : group I(N=43,0%-50%), group II(N=49, 50%-150%) and group III(N=44, 150%-200%). To evaluate the effects of periovarian adhesions on follicular development in controlled ovarian yperstimulation for IVF-ET, serum E2 levels on the day of hCG dministration (Day 0) and the day after hCG administration (Day+1), the number of ovarian follicles with mean diameter${\geqq}$12mm on Day 0, and the number of oocytes retrieved by transvaginal aspiration were measured and compared among groups. There were no significant differences in age of patients, cancellation rate due to inadequate ovarian response, serum E2 levels, the number of ovarian follicles, the number of oocytes retrieved, and oocytes retrieval rate per follicle. In the same patients(N=31) in group II in whom the difference in ovarian availability between two ovaries is more than 50%, there was also no significant difference in the number of ovarian follicles between them. These data suggest that pelvic adhesions including periovarian adhesions have no adverse effects on the ovarian response to gonadotropins stimulation and the outcome of IVF-ET.