Ovarian Strips Research Articles

Facilitation of Ovarian Response by Mechanical Force-Latest Insight on Fertility Improvement in Women with Poor Ovarian Response or Primary Ovarian Insufficiency.

The decline in fertility in aging women, especially those with poor ovarian response (POR) or primary ovarian insufficiency (POI), is a major concern for modern IVF centers. Fertility treatments have traditionally relied on gonadotropin- and steroid-hormone-based IVF practices, but these methods have limitations, especially for women with aging ovaries. Researchers have been motivated to explore alternative approaches. Ovarian aging is a complicated process, and the deterioration of oocytes, follicular cells, the extracellular matrix (ECM), and the stromal compartment can all contribute to declining fertility. Adjunct interventions that involve the use of hormones, steroids, and cofactors and gamete engineering are two major research areas aimed to improve fertility in aging women. Additionally, mechanical procedures including the In Vitro Activation (IVA) procedure, which combines pharmacological activators and fragmentation of ovarian strips, and the Whole Ovary Laparoscopic Incision (WOLI) procedure that solely relies on mechanical manipulation in vivo have shown promising results in improving follicle growth and fertility in women with POR and POI. Advances in the use of mechanical procedures have brought exciting opportunities to improve fertility outcomes in aging women with POR or POI. While the lack of a comprehensive understanding of the molecular mechanisms that lead to fertility decline in aging women remains a major challenge for further improvement of mechanical-manipulation-based approaches, recent progress has provided a better view of how these procedures promote folliculogenesis in the fibrotic and avascular aging ovaries. In this review, we first provide a brief overview of the potential mechanisms that contribute to ovarian aging in POI and POR patients, followed by a discussion of measures that aim to improve ovarian folliculogenesis in aging women. At last, we discuss the likely mechanisms that contribute to the outcomes of IVA and WOLI procedures and potential future directions.

P-456 Differential distribution of the extracellular matrix components within the human ovarian cortex and remodelling during in vitro culture

Abstract Study question How is the extracellular matrix (ECM) network distributed within the human ovarian cortex, and how does in vitro culture alter its structure? Summary answer The ECM composition varies in the different layers of the ovarian cortex and is further remodelled during in vitro culture. What is known already The ovarian ECM is the scaffold within which follicles and stromal cells are organised. Its composition and structural properties constantly evolve to accommodate follicle development and expansion. Culturing primordial follicles within ovarian strips enables to reproduce the exact composition and stiffness of the native ECM. Yet, tissue preparation, which involves mechanical loosening, induces modifications in the ECM network and alters cell-cell contact, leading to spontaneous follicle activation. Characterising the native ovarian cortical ECM and its dynamic changes during culture will help decipher its role during folliculogenesis and improve both in vitro activation (IVA) and in vitro growth (IVG) systems. Study design, size, duration Fresh ovarian cortical biopsies were obtained from 6 women aged 28–38 years (mean ± SD: 32.7 ± 4.1 years) at elective caesarean section. Biopsies were cut into fragments of ∼4 × 1×0.5 mm and cultured for 0, 2, 4 or 6 days. Participants/materials, setting, methods Stromal cell density as well as the percentage of ECM-related protein (collagen, elastin, fibronectin, laminin) positive area in the entire cortex were quantified at each time point using histological and immunohistological analysis. Collagen and elastin contents were further quantified within each layer of the human ovarian cortex, namely the outer cortex, the mid-cortex, and the cortex-medulla junction regions. Main results and the role of chance Collagen content of the cortical ECM decreased from 55.5% ± 1.7% positive area at day 0 (D0) to 42.3% ± 1.1% at D6 (p = 0.001), and elastin increased from 1.1% ± 0.2% at D0 to 1.9% ± 0.1% at D6 (p = 0.001). Fibronectin and laminin remained stable. This suggests tissue loosening during culture, in accordance with the decreased stromal cell density from 3.6x106 ± 0.6 to 2.8x106 ± 0.3 cells/mm3 at D2 (p = 0.033) with no subsequent change. Moreover, collagen and elastin distribution were uneven throughout the cortex and during culture. Collagen deposition was maximal at the outer cortex and the lowest at the mid-cortex (69.4% ± 1.6% vs 53.8% ± 1.5% positive area, p < 0.001), and decreased from D0 to D2 (65.2% ± 2.0% vs 60.6% ± 2.2%, p = 0.033) then stabilised. Elastin showed the converse distribution, being most concentrated at the cortex-medulla junction (3.7% ± 0.3% vs 0.9% ± 0.2% in the outer cortex, p < 0.001), and peaked at D6 compared to D0 (3.1% ± 0.3% vs 1.3% ± 0.2%, p < 0.001). These data indicate a distinct phenotype of the ovarian cortical ECM depending on both its region and the culture period. Further characterisation of the differences in ECM architecture is ongoing. Limitations, reasons for caution Ovarian cortical biopsies were obtained from pregnant women undergoing caesarean sections. As such, the data obtained may not accurately reflect the ECM distribution of non-pregnant women. Wider implications of the findings Clarifying the composition and architecture signature of the ovarian cortical ECM will not only provide a foundation for further exploration of ovarian microenvironments, but also be of importance for understanding of ECM-follicle interactions maintaining the primordial pool and early growth stages leading to improvements in IVA and IVG. Trial registration number not applicable

Mixed Gonadal Dysgenesis in a Mosaic 45,X / 46,X+Mar (with +SRY/TPSY/DYZ3 genes): A Rare Case Report

Introduction Y chromosome-derived material in Turner´s Syndrome patients is a risk factor for the malignant transformation of the dysgenetic gonad and/or virilization. Small fragments of Y chromosome, can be missed by conventional cytogenetics, so PCR techniques for such markers can aid in the identification. Study Objective To present a rare case of Gonadal Dysgenesis. Design Video case report. Setting Laparoscopic approach and complete genetic and histopathological study. Patients or Participants 14-year-old female patient with primary amenorrhoea complained after 1 year of clitoris enlargement and deepening of the voice. She had short height, low posterior hairline with low set ears, short neck, cubitus valgus and incipient bilateral breast buds. External gynecologic was unremarkable for an enlarged clítoris. Her karyotype was Mos45,X/46,X,+mar. FISH for Y Chromosome was +, and molecular determination through PCR for specific Y chromosome genes SRY, TSPY and DYZ3 were also +. Lab tests: free testosterone 128 ng/dl (normal 15-70 ng/dl), a normal DHEA-S (64 mcg/dl) and androstenodione (0.5ng/ml). Interventions She was counseled for laparoscopic gonadectomy. Measurements and Main Results Uterus was hypoplastic. Her left ovary was a 4 cm cystic appearance opaque smooth mass of unusual appearance for a normal ovary. Her right ovary was an underdeveloped ovarian strip. She was discharged on POD 1 Her biopsy showed normal testicular stroma with seminiferous tubules and hyperplasic Leydig cells on the left gonad and normal ovarian tissue on the right gonad. Conclusion Of all Turner´s Syndrome patients, only a 2-5% will have a mosaic 45,X/46,XY karyotype. Any TS patient with signs of virilization must be investigated for the presence of Y chromosome genes. If SRY or similar genes are present, surgical removal of the dysgenetic gonads must be done in order to treat virilization and prevent malignant transformation. Y chromosome-derived material in Turner´s Syndrome patients is a risk factor for the malignant transformation of the dysgenetic gonad and/or virilization. Small fragments of Y chromosome, can be missed by conventional cytogenetics, so PCR techniques for such markers can aid in the identification. To present a rare case of Gonadal Dysgenesis. Video case report. Laparoscopic approach and complete genetic and histopathological study. 14-year-old female patient with primary amenorrhoea complained after 1 year of clitoris enlargement and deepening of the voice. She had short height, low posterior hairline with low set ears, short neck, cubitus valgus and incipient bilateral breast buds. External gynecologic was unremarkable for an enlarged clítoris. Her karyotype was Mos45,X/46,X,+mar. FISH for Y Chromosome was +, and molecular determination through PCR for specific Y chromosome genes SRY, TSPY and DYZ3 were also +. Lab tests: free testosterone 128 ng/dl (normal 15-70 ng/dl), a normal DHEA-S (64 mcg/dl) and androstenodione (0.5ng/ml). She was counseled for laparoscopic gonadectomy. Uterus was hypoplastic. Her left ovary was a 4 cm cystic appearance opaque smooth mass of unusual appearance for a normal ovary. Her right ovary was an underdeveloped ovarian strip. She was discharged on POD 1 Her biopsy showed normal testicular stroma with seminiferous tubules and hyperplasic Leydig cells on the left gonad and normal ovarian tissue on the right gonad. Of all Turner´s Syndrome patients, only a 2-5% will have a mosaic 45,X/46,XY karyotype. Any TS patient with signs of virilization must be investigated for the presence of Y chromosome genes. If SRY or similar genes are present, surgical removal of the dysgenetic gonads must be done in order to treat virilization and prevent malignant transformation.

Autotransplantation of the ovarian cortex after in-vitro activation for infertility treatment: a shortened procedure.

Is it possible to establish a new in-vitro activation (IVA) protocol for infertility treatment? A new IVA procedure is an efficient and easily performed approach for infertility treatment of patients with diminished ovarian reserve (DOR). IVA of primordial follicles with or without stimulators has been developed to treat patients with primary ovarian insufficiency (POI) successfully. However, the efficiency of the procedure is still very low. There is a requirement to optimize the protocol with increased efficiency for clinical application. Newborn mouse ovaries were used to establish a new 1-h IVA protocol with the mechanistic target of rapamycin (mTOR) stimulator phosphatidic acid (PA, 200 µM) and the phosphatidylinositol-3-kinase (PI3K) stimulator 740Y-P (250 µg/ml); a prospective observational cohort study in POI patients was performed on 15 POI patients and 3 poor ovarian response (POR) patients in three different centers of reproductive medicine in China. One-third of ovarian cortex was removed and processed into bigger strips (1 × 1 cm2, 1-2 mm thickness). Strips were then sutured back after treatment. The new approach only requires one laparoscopic surgery. Follicular activation and development increased in cultured mouse and human ovarian tissues after 1 h of stimulator treatment. Compared with tiny ovarian cortex pieces (1 × 1 mm2), large ovarian strips (1 × 1 cm2) showed the lowest apoptotic signals after incubation. We applied the orthotropic transplantation procedure with large strips in the clinic, and 9 of 15 POI patients showed at least one-wave follicular growth during the monitoring period. One patient was reported with one healthy delivery after natural conception and another patient with a healthy singleton delivery after IVF. All the contacted patients (n = 13) responded with no side effects on their health 2-4 years after IVA procedure. Further clinical trials with a large number of well-defined patients are required to compare different IVA protocols. A long-term follow-up system should be set up to monitor patient's health in the future cohort study. By using stimulators, the findings in the study provide a more efficient IVA protocol for the treatment of patients with DOR. It requires only one laparoscopic surgery and thus minimizes patients' discomfort and costs. This strategy could be useful for patients diagnosed with POI and desire pregnancy as soon as possible after the operation. This work was supported by the National Key Research and Development Program of China (2018YFC1003703 and 2018YFC1004203); the National Natural Science Foundation of China (81871221); Co-construction of Provincial Department (201601006). The authors have no conflict of interest to disclose. ChiCTR2000030872.

Epinephrine minimizes the use of bipolar coagulation and preserves ovarian reserve in laparoscopic ovarian cystectomy: a randomized controlled trial

We propose a novel method, the epinephrine compression method (Epi-pledget), as a hemostasis method for ovarian cystectomy. A total of 179 patients undergoing laparoscopic ovarian cystectomy with stripping were randomly allocated into three groups: the bipolar coagulation group, the Epi-pledget group, and the coagulation after Epi-pledget (Epi & Coagulation) group. Serum anti-Müllerian hormone (AMH) levels and antral follicle count (AFC) by ultrasonography were measured to determine the preservation of ovarian function. To evaluate the postoperative ovarian cellular proliferative activity and tissue damage in a mouse model, we operated on the ovaries of mice with an artificial incision injury and applied two hemostatic methods: coagulation and Epi-pledget. Eight weeks after surgery, the AMH rate significantly decreased in the bipolar coagulation group compared with the Epi-pledget group. The AFC decline rate was also significantly greater in the coagulation group than the Epi-pledget group. Specifically, patients with endometrioma had a significantly greater decline of serum AMH in the coagulation group than the Epi-pledget group. In a histopathological analysis in mice, the Epi-pledget group showed ameliorated fibrotic changes and necrotic findings in the injured lesion compared with the bipolar coagulation group. The Epi-pledget method for ovarian stripping has an additional benefit of maximizing the preservation of the ovarian reserve, especially for the endometriotic ovarian cyst type.

Primordial follicle survival of goat ovarian tissue after vitrification and transplantation on chorioallanthoic membrane

BackgroundOvarian tissue vitrification is an alternative fertility preservation procedure for young female patients prior to gonadotoxic treatment. Primordial follicle loss might be a potential issue for vitrification and transplantation procedures. This study aimed to evaluate primordial follicle density and deoxyribonucleic acid (DNA) fragmentation in each stage of the preservation procedure of goat ovarian tissue. Follicle density and DNA fragmentation were examined microscopically after staining with hematoxylin eosin and TUNEL assay, respectively. Both parameters were compared between fresh, fresh-transplanted, vitrification, and vitrification-transplanted groups.ResultsA significant decrease was observed in the primordial follicle proportion after vitrification and transplantation compared to the primordial follicle proportion in the fresh group (88.09% vs 52.42%, p < 0.05, 95% CI 11.54, 66.94). There was no significant difference in DNA fragmentations of primordial follicles between each group (p > 0.05).ConclusionsThe vitrification and transplantation process of goat ovarian strips could cause the primordial follicles loss and DNA damage of the follicles. However, primordial follicles loss and DNA damage were not significantly different in each procedure.

Slush nitrogen vitrification of human ovarian tissue does not alter gene expression and improves follicle health and progression in long-term in vitro culture

To study whether slush nitrogen (SN) vs. liquid nitrogen (LN) vitrification affects human ovarian tissue gene expression and preserves follicle health during extended invitro culture. Randomized experimental study. University research laboratory. Ovarian biopsies collected by laparoscopic surgery from patients with benign gynaecologic conditions. None. Ovarian strips were vitrified with LN or SN, warmed, and analyzed before or after culture for 9days (d9) in gas-permeable dishes. Expression of genes involved in stress and toxicity pathways was analyzed in fresh and warmed strips by polymerase chain reaction (PCR) array and quantitative real-time-PCR. Fresh and vitrified/warmed strips were analyzed for follicle quality, progression, and viability before or after culture. The SN vitrification preserved follicle quality better than LN (% grade 1 follicles: fresh control, 54.2; LN, 29.3; SN, 48.8). Quantitative reverse transcription-PCR demonstrated a noticeable up-regulation of 13 genes in LN samples (range, 10-35) and a markedly lower up-regulation of only 5 genes (range, 3.6-7.8) in SN samples. Long-term invitro culture evidenced worse follicle quality and viability in LN samples than in both fresh and SN samples (% grade 1 follicle: fresh d0, 51.5; fresh d9, 41; LN d9, 16.4; SN d9, 55) and a highly significant reduction of primordial follicles and a concomitant increase of primary and secondary follicles in all samples. Follicle growth to the secondary stage was significantly higher in vitrified tissue than in fresh tissue, being better in SN than in LN vitrified tissue. Follicle quality, gene expression, viability, and progression are better preserved after SN vitrification.

Is oxygen availability a limiting factor for in vitro folliculogenesis?

Transplantation of ovarian tissue for the preservation of fertility in oncological patients is becoming an accepted clinical practice. However, the risk of re-introducing tumour cells at transplantation has stirred an increased interest for complete in vitro folliculogenesis. This has not yet been achieved in humans possibly for the lack of knowledge on the environmental milieu that orchestrates folliculogenesis in vivo. The main aim of this study was to investigate the effect of oxygen availability on follicle health and growth during in vitro culture of ovarian tissue strips. To this end, a model was developed to predict the dissolved oxygen concentration in tissue under varying culture conditions. Ovarian cortical strips of bovine, adopted as an animal model, and human tissue were cultured in conventional (CD) and gas permeable (PD) dishes under different media column heights and gaseous oxygen tensions for 3, 6 and 9 days. Follicle quality, activation of primordial follicles to the primary stage, and progression to the secondary stage were analysed through histology. Follicle viability was assessed through a live-dead assay at the confocal scanning laser microscope. Findings showed a higher follicle quality and viability after culture of bovine ovarian strips in PD in adequate medium height and oxygen tensions. The best culture conditions found in the bovine were adopted for human ovarian strip culture and promoted a higher follicle quality, viability and progression. Overall, data demonstrated that modulation of oxygen availability in tissue plays a key role in maintaining follicles’ health and their ability to survive and progress to the secondary stage during ovarian tissue in vitro culture. Such culture conditions could increase the yield of healthy secondary follicles for subsequent dissection and individual culture to obtain competent oocytes.

126 EFFECTIVE METHOD FOR IN VITRO CULTURE OF CRYOPRESERVED OVINE OVARIAN TISSUE

Today, ovarian tissue cryopreservation is used for preserving the reproductive function of women, as well as the genetic material of rare and endangered species or domestic animals breeds. In the last 20 years genetic diversity of farm animals breeds suffered considerable losses in Kazakhstan; therefore, genetic preservation of valuable local breeds is desirable. The aim of this study was to compare the effectiveness of different in vitro culture media on morphology of ovine ovarian tissue cryopreserved by a slow-freezing protocol with 1.5 M dimethyl sulfoxide (DMSO). Ovaries were collected from indigenous Chuyi breed and immediately transported to the laboratory at 30°C within 1 h. Ovaries were rinsed several times in PBS supplemented with antibiotics (75 mg L–1 of penicillin-G, 50 mg L–1 of streptomycin sulfate). In Hepes-buffered medium 199, halved and the medulla removed with curved iris. Using a scalpel, the cortex was cut into 5- × 3- × 1-mm strips. Ovarian strips were equilibrated sequentially in freezing medium containing 0.25, 0.75, and 1.5 M DMSO with 0.5 M sucrose (5 min each). Then, ovarian strips were frozen in plastic straws using a programmable freezer Planer Kryo-360 3,3 (Planer, UK) and cooled as follows: stabilised at 20°C for 5 min, cooled from 20°C to –70°C at 5°C min–1, seeded to the temperature –7°C, cooled again to –30°C at 0.3°C min–1, cooled to –150°C at 35°C min–1, and finally plunged into liquid nitrogen and stored for 10 days. The straws were thawed at room temperature for 1 min, and then immersed in a water bath at 37°C for 2 min, warmed at room temperature with Dulbecco’s PBS (DPBS), supplemented with 10% FCS and 0.75 M sucrose (15 min), then DPBS + 10% FCS (30 min), and finally placed in the culture media for 10 min. Fresh and frozen tissue pieces were randomly distributed into 12 groups for further culture: 1) TCM 199 + 10% FBS; 2) TCM 199 + 10% native ovine serum (NOS); 3) TCM-Hepes + 10% FBS; 4) TCM-Hepes + 10% NOS; 5) DMEM + 10% FBS; 6) DMEM + 10% NOS; with and without 7.5 mg mL–1 of FSH. After 7 days of culture, the effects of different culture media on ovarian tissue morphology was evaluated by light microscopy after hematoxylin and eosin staining of tissue sections. The best result was observed when frozen ovarian tissue was cultured in the presence of FSH. The best result was observed in group 3 and 4 with FSH. The percentages of normal primordial, primary, and preantral follicles were: 1) TCM 199 + 10% FBS + FSH = 53.5 ± 3.1, 39.7 ± 3.8a, 28.5 ± 3.2; 2) TCM 199 + 10% NOS + FSH = 49.4 ± 2.3a, 36.7 ± 3.3a, 25.3 ± 4.1; 3) TCM-Hepes + 10% FBS + FSH = 66.3 ± 2.5, 45.7 ± 3.9, 35.1 ± 3.8; 4) TCM-Hepes + 10% NOS + FSH = 86.5 ± 3.8b, 75.4 ± 4.2b, 45.7 ± 3.5; 5) DMEM + 10% FBS + FSH = 42.1 ± 3.5a, 33.7 ± 2.9a, 21.3 ± 4.9, 20.7 ± 3.9; 6) DMEM + 10% NOS + FSH = 41.3 ± 3.9a, 32.9 ± 2.5a; control group = 98.2 ± 1.1, 93.7 ± 1.7, 90.3 ± 1.9 (ab, P &lt; 0.01). The majority of follicles in groups without FSH were degenerated. In group 4) TCM-Hepes + 10% NOS without FSH, a damaged structure of primordial follicles was observed.

Ovarian performance, IVF results, pregnancies and live births indicate; fertility preservation using ovarian tissue harvesting and transplantation of thawed ovarian strips is effective

The field of fertility preservation (FP) in females has extended with increasing medical and public awareness. Significant scientific achievements provided solid proofs indicating that female fertility can be preserved over time. A commonly practiced technique for FP is ovarian tissue cryopreservation, however, there is shortage of publications describing clinical experience, success rate and outcomes data of this procedure.

Ovarian stripping versus cystectomy: recurrence of endometriosis and pregnancy rate

To compare two different surgical techniques, stripping or cystectomy, in patients treated with the same post-operative medical therapy in terms of recurrence of endometrioma, recurrence of pain and spontaneous pregnancy rate within 2 years from surgery. The inclusion criteria of this study were: (1) 25-40 years old; (2) ovarian endometrioma more than 3 cm of diameter detected by transvaginal ultrasonography (3) regular menstrual cycle (4) post-operative treatment with GnRH analogs, (5) tubal patency assessed by laparoscopic chromopertubation (6) normal human semen characteristics. Exclusion criteria were uterine myoma, previous medical treatment for endometriosis, presence of adenomyosis, previous surgery of ovarian endometrioma, multiple cysts, bilateral involvement, co-existence of deep endometriosis. Patients were assigned to two study groups: group A (N = 45) patients undergoing stripping technique and group B (N = 64) patients undergoing cystectomy technique for ovarian endometrioma. In group B the percentage of ultrasonographic recurrence (15.4 %, N = 15) is much lower than in group A (55.6 %, N = 25). (p value 0.001). In group B the percentage of symptomatic recurrence (21.8 %, N = 14) is much lower than in group A (53.3 %, N = 24) (p value 0.001). Spontaneous pregnancy rate in group A patients was of 4.4 % (N = 2) and in group B 22.3 % (N = 21), (p value 0.0072). However, the percentage of specimen with adjacent healthy ovarian tissue was lower in group A (26.6 %) than in group B (50 %) (p value 0.01). Among the different treatment options for surgical treatment of ovarian endometrioma, in our experience cystectomy appears to be the most appropriate treatment, both in terms of recurrence and pregnancy rate.

In vitro development of human primordial follicles to preantral stage after vitrification

The aim was to culture primordial follicles in vitro to reach preantral stage in vitrified human ovarian tissue. Ovarian tissue samples were obtained from six women. Tissue strips were vitrified by infiltration with a cryoprotectant followed by mounting on a stainless steel carrier. After culturing for 7 days the morphology and developmental stages of follicles enclosed in fresh and vitrified groups were analyzed. High proportion of viable follicles in vitrified ovarian strips was obtained. After culturing for 7 days the percentage of secondary and preantral follicles increased significantly (P < 0.05) whereas primordial and transitory follicles showed a significant decrease (P < 0.05) compared to their respective counterparts at day 0 of culture. Vitrification of ovarian strips with an improved carrier device and culturing of follicles in ovarian strips after warming yielded developed follicles with high viability and morphological integrity that may be suitable for use in fertility preservation among cancer patients.

Optimal condition of vitrification method for cryopreservation of human ovarian cortical tissues

In order to find the optimal exposure time of cryoprotectant, we performed a comparison of vitrification versus slow freezing according to the degree of normal morphology and apoptosis of human ovarian follicles. Eleven patients aged 20-41 years who underwent operative laparoscopy for benign ovarian cysts or cesarean section were enrolled in this study. We carried out a prospective parallel comparison of survival and morphology of follicles after freezing (slow freezing and vitrification) and thawing. The ovarian strips were vitrified with two-step exposure to equilibration and vitrification solutions at room temperature. After various exposure times of cryoprotectant solution (5 min, 10 min, and 20 min, respectively), cryoprotectant-filled cryovials with pretreated cortical tissues were immediately plunged into liquid nitrogen. In total, 336 follicles were analyzed by light microscopy to assess the morphology. The distribution of follicles was as follows: primordial, primary, and secondary follicles were 55.7% (187/336), 36.9% (124/336), and 7.4% (25/336), respectively. Vitrification in the 10-min exposure group preserved the follicles most effectively (ratio of grade 1 follicle: 3.6%, 34.7%, 13.8%, and 20.0% in the 5-min, 10-min, 20-min, and slow-freezing groups, respectively). Fewer terminal-deoxynucleotidyl-transferase-dUTP-nick-end-labeling-positive cells were found in vitrification in the 10-min equilibrium group compared with the other cryopreserved-thawed groups (52.1%, 31.5%, 53.1%, and 46.7% in the 5-min, 10-min, 20-min, and slow-freezing groups, respectively). The stromal cells were also better preserved in the 10-min group than the others (P < 0.05). The 10-min exposure group for vitrification showed better results compared with other conditions and the slow-freezing group.

Effect of KIT Ligand on the Viability of Porcine Primordial Follicles in Vitro

Abstract: In mammals, the mechanisms regulating the initiation of follicle development remain poorly understood. We previously reported that testosterone induced the activation of porcine primordial follicles after 7 days of culture. Herein, the effect of KIT ligand (KL) on the viability, activation and development of porcine primordial follicles was examined. Ovarian strips containing primordial follicles were cultured for 7 days in a medium supplemented with KL (10, 100, 250 or 500 ng/ml). The percentage of degenerated follicles was significantly decreased (39 ± 2%) in the 100 ng/ml KL-treated group compared to the control (56 ± 1%), whereas almost all of the primordial follicles degenerated at higher concentrations of KL. No primordial follicles developed in culture at any concentration of KL. When ovarian strips were cultured in medium supplemented with testosterone (10-6 M) + KL (100 ng/ml), primordial follicles developed to early secondary follicles (5 ± 3%), however the total percentages of develop...

ENDOTHELIN-2 (EDN2) INDUCED OVARIAN AND OVIDUCTAL CONSTRICTION VIA ENDOTHELIN RECEPTOR A (ETA) IN RATS

In the rat, expression of mRNA for END2 in granulosa cells dramatically, yet transiently, increases at the time of ovulation and antagonism of EDN2 receptor binding blocks ovulation. Because EDN2 is a potent constrictor of smooth muscle cells these results suggest that EDN2 may be a final contractile signal regulating ovulation. Our first objective was to further examine the EDN2 / receptor system in the ovary at the time of ovulation. In all experiments, follicular development was induced in immature rats by treatment with PMSG (10 IU) and the ovulatory cascade was induced 48 h later by treatment with hCG (10 IU). In Exp. 1, total ovarian concentrations of endothelin were determined at 0 and 12 h after hCG by enzyme-immunoassay. Ovarian levels of endothelin peptide were increased 7-fold at 12 h after treatment with hCG (P < 0.05), demonstrating that EDN2 peptide is produced prior to ovulation. In Exp. 2, isometric tension analysis was conducted using strips of ovarian tissue collected from PMSG/hCG primed rats at 10 h after hCG to determine which endothelin receptor subtype (ETA or ETB) is mediating the action of EDN2. An ETA (BQ123), but not ETB (BQ788), receptor antagonist inhibited EDN2-induced constriction of ovarian strips (P < 0.05), suggesting that EDN2 acts via ETA at ovulation. Our next objective was to examine the EDN2 / receptor system in the oviduct. In Exp. 3, cumulus-oocyte complexes (COCs) were isolated from the ovary 48 h after PMSG and at 12 h after hCG, and from the oviduct at 20 h after hCG. Real-time PCR analysis demonstrated the dramatic induction of EDN2 mRNA in COCs collected at 12 h after hCG (P < 0.05) but also revealed an 11-fold increase in expression of EDN2 mRNA in COCs collected at 20 h after hCG, compared to 48 h after PMSG (P < 0.05), suggesting that EDN2 produced from the ovary at ovulation and/or by the COCs within the oviduct may affect oviductal contractility and oocyte transport. In Exp. 4, isometric tension analysis using sections of the oviduct was conducted to determine whether the oviduct responds to EDN2 and if so, by which receptor subtype this effect was mediated through. Treatment with EDN2 induced a potent constriction of oviductal sections (P < 0.05) and concurrent treatment with BQ123, but not BQ788, inhibited EDN2- induced constriction. Immunohistochemical examination of sections of oviductal tissue also revealed strong staining for ETA but not ETB in the luminal mucosa of the oviduct. In conclusion, EDN2 is produced by granulosa cells at ovulation and appears to be acting through the ETA receptor subtype. EDN2 may also be produced by ovulated COCs within the oviduct. Contractile tissue in both the ovary and oviduct responded to treatment with EDN2, suggesting that in addition to the requirement for EDN2 at ovulation, EDN2-induced oviductal constriction may affect transportation of COCs to the site of fertilization. Supported by NIH P20 RR15592 and RO1 HD052684. (poster)

Two novel techniques to detect follicles in human ovarian cortical tissue

Ovarian tissue cryopreservation and transplantation are becoming increasingly important issues for preserving female fertility as shown by recent successes in restoring ovarian activity and even fertility. Primordial follicle content before transplantation is a key issue for success. We investigated two novel methods to detect primordial follicles in human ovarian cortical tissue strips. The first method used the fluorescent mitochondrial stain rhodamine 123 in combination with laser scanning confocal microscopy (LSCM). The first method used the fluorescent mitochondrial stain rhodamine 123 (R123) in combination with laser scanning confocal microscopy (LSCM). The second used a simple stereomicroscopic method with glass-bottom dishes for detecting primordial follicles in ovarian cortical tissue strips. Potential toxic effects of R123 and of the exposure to confocal laser were investigated in a mouse ovarian allograft model. Follicles were visible as white spots in thin cortical strips using LSCM in single and fast scanning at low magnification, allowing a fair estimation of the number of primordial follicles present. Using the second method, ovarian follicles were also visible using glass-bottom dishes under the stereomicroscope, although tissue thickness and density were limiting factors of its success. Follicles can be visualized in human cortical ovarian strips with R123 in combination with LSCM. Stereomicroscopy using glass-bottom dishes and transmitted illumination is a simple alternative method and has the advantage of allowing further safe clinical use of the analysed tissue.

Ovarian Tissue Storing for Fertility Preservation in Clinical Practice: Successful Pregnancy, Technology, and Risk Assessment

Cryopreservation of ovarian tissue has been experimentally practiced during the last decade in an attempt to preserve fertility, mostly in cancer patients before administration of chemotherapy. In order to establish the procedure in clinical practice a successful transplantation of cryopreserved thawed tissue- as indicated by fertility restoration and a normal pregnancy- should be demonstrated. A simple transplantation technique that allows conventional infertility treatments or natural conception, and methods that maximize the safety of the procedure and prevent cancer cells transplantation are indicated. We report of 2 patients who requested transplantation of stored ovarian tissue in order to overcome ovarian failure post bone marrow transplantation. Ovarian tissue was harvested from 150 cancer patients by laparoscopic procedure before administration of high dose chemotherapy, in a tertiary medical center. Two young patients, suffering from ovarian failure post therapy requested transplantation of stored ovarian tissue in order to overcome infertility. One was treated for Chronic Myeloid Leukemia (CML) (age 25), and the 2nd suffered from Non Hodgkin Lymphoma (NHL) (age 30). Minimal disease detection:In both patients small frozen fragments (separately kept) were initially thawed for detection of possible malignant cells and assessing the quality of follicles post thawing. For the CML patient real time RT-PCR for bcr-abl m-RNA indicating the presence of Philadelphia chromosome was performed. There were no molecular markers for the NHL patient. Transplantation technique:By laparotomy, three pairs of 5mm transverse incisions were made through the tunica albuginea, and using blunt dissection, cavities were formed beneath the cortex, into which three thawed ovarian strips (0.5X1.5cm each) were gently placed and sutured with 4/0 Vicryl. PCR product was detected in the ovarian tissue of the CML patient, thus the tissue was not transplanted. Following transplantation to the NHL patient endocrine studies (gonadotrophins, ovarian steroids, inhibin B and AMH) indicated full recovery 8 months post transplantation. An IVF cycle was performed using controlled stimulation cycle followed by egg retrieval using conventional setting for follicle aspiration. A mature egg was aspirated fertilized and a four-cell embryo was transplanted (48h). A normal pregnancy, currently at third trimester is monitored 1. In order to increase the safety of ovarian tissue cryopreservation-transplantation procedure and reduce the risk of transferring malignant cells back to cured cancer patients it is mandatory to use the best methods to detect microscopic ovarian metastases. 2. This is the first report of oocyte retrieval, IVF and pregnancy in a sterilized patient following transplantation of cryopreserved-thawed ovarian tissue. The pregnancy and production of ovarian hormones provided strong evidence for the recovery of the transplant and confirmed the quality and fertility potential of cryopreserved thawed ovarian tissue. Fertility preservation with ovarian cortical tissue storing is effective in humans and the ovary is a good location for transplantation since conventional fertility procedures can be used.

Laparoscopic removal of endometriomas: sonographic evaluation of residual functioning ovarian tissue

Objective The aim of this study was to determine whether and to what extent laparoscopic removal of ovarian endometriotic cysts is a tissue-sparing procedure. Study design At the University Hospital, 77 women of reproductive age with endometriomas and 55 with dermoid cysts underwent laparoscopic removal of the ovarian disease by stripping. Within 1 month before and within 36 months after surgery all patients underwent transvaginal sonographic evaluation of ovarian volume of the endometriomas or dermoid cysts and measurement of the residual ovarian tissue. Results The residual ovarian volume after surgery was significantly less for the endometrioma group than for the dermoid group. Comparison of the volume of the treated ovary with that of the untreated contralateral ovary showed a significant difference (4.3±2.3 cm 3 vs 9.7±3.9 cm 3) only in the endometrioma group. Conclusion Ovarian stripping of endometriomas, but not of ovarian dermoids, is associated with a significant decrease in residual ovarian volume which may result in diminished ovarian reserve and function.

Results of second-look laparoscopy following removal of endometriomata

Despite widespread use of operative laparoscopy for removal of endometriomata, there are inadequate data regarding subsequent adhesion formation. We performed second look laparoscopy (SLL) within 6 months of laparoscopic endometriosis cystectomy in 21 infertile women. Mean age and mean duration of infertility were 30.5 &plusmn; 4.4 and 7.1 &plusmn; 4.9 years respectively. Cysts were removed with the following technique: incision of the ovarian cortex, cystoscopy (biopsy when necessary), and total stripping of the cyst capsule. All patients received postoperative suppressive therapy for 3 months. Total AFS score decreased significantly in all patients (34.1 &plusmn; 14.7 vs 12.3 &plusmn; 10.7; P<0.001). The adhesion score assessed at SLL improved in 3 (14.3%) remained the same in 13 (61.9%) and worsened in 5 (23.8%). Mean adhesion score at SLL was significantly less compared with adhesion score during cystectomy (7.1 &plusmn; 5.2 vs 8.7 &plusmn; 6.4; P=0.038). None of the patients had de novo adhesions. Adhesion scores were similar in women where ovarian cortical defect was left open or suture closed. Of the 21 women, 4 (19%) conceived within six months of SLL. In conclusion, laparoscopic removal of ovarian endometriomata was not associated with increased adhesion formation at SLL. Adhesions at SLL were mainly dependent upon adhesions at laparoscopic cystectomy.