Learning Curve of Surgeons Performing Laparoscopic Ovarian Tissue Transplantation in Women with Premature Ovarian Insufficiency: A Statistical Process Control Analysis.

STUDY OBJECTIVE: To analyze patient safety in laparoscopic ovarian tissue transplantation surgery by tracking the rate of postoperative complications and the learning curves of the surgeons by statistical process control analysis. DESIGN: A retrospective study. SETTING: A university-affiliated hospital. PATIENTS: A total of 100 patients with premature ovarian insufficiency who underwent ovarian tissue cryopreservation by vitrification and then autologous transplantation of frozen-thawed ovarian tissues with in vitro activation. INTERVENTIONS: Ovarian tissue cryopreservation, in vitro activation, and transplantation. MEASUREMENTS AND MAIN RESULTS: We assessed the surgery complications, differences in total surgery time, transplantation time, and transplantation time per ovarian sheet in operations performed by 3 experienced laparoscopic surgeons. Surgeon A performed 80 operations; surgeon B, 29 operations; and surgeon C, 20 operations. Complications occurred in 1.55% of the procedures. Although all 3 surgeons' performance never fell below the unacceptable failure limit, only surgeon A became competent after 66 cases. CONCLUSION: The laparoscopic ovarian tissue transplantation surgery was generally safe given that the postoperative complications were infrequent (1.55%). Although the performance of all 3 surgeons was acceptable, only surgeon A attained the level of competency after 66 cases. The transplantation method may not be the key factor for reducing surgery time in this surgery. An efficient ovarian tissue transplantation team is more important in reducing the surgery time than the surgeon's surgical technique alone.

Successful fertility preservation following ovarian tissue vitrification in patients with primary ovarian insufficiency.

STUDY QUESTION: Is ovarian tissue cryopreservation using vitrification followed by in vitro activation (IVA) of dormant follicles a potential approach for infertility treatment of patients with primary ovarian insufficiency (POI)? SUMMARY ANSWER: Our vitrification approach followed by IVA treatment is a potential infertility therapy for POI patients whose ovaries contain residual follicles. WHAT IS KNOWN ALREADY: Akt (protein kinase B) stimulators [PTEN (phosphatase with TENsin homology deleted in chromosome 10) inhibitor and phosphatidyinositol-3-kinase (PI3 kinase) stimulator] activate dormant primordial follicles in vitro and ovarian fragmentation disrupts the Hippo signaling pathway, leading to the promotion of follicle growth. We treated POI patients with a combination of ovarian vitrification, fragmentation and drug treatment, followed by auto-transplantation, and reported successful follicle growth and pregnancies. STUDY DESIGN, SIZE, DURATION: Prospective clinical study of 37 infertile women with POI between 12 August 2011 and 1 November 2013. We enrolled 10 new patients since the previous publication. PARTICIPANTS/MATERIALS, SETTING, METHODS: POI patients were originally selected based on a history of amenorrhea for more than 1 year and elevated serum FSH levels of >40 mIU/ml (n = 31) but this was later changed to >4 months, age <40 years and serum FSH levels of >35 mIU/ml (n = 6) (mean 71.8 ± 30.8, range 35.5-197.6) so as to include patients with a shorter duration of amenorrhea. Under laparoscopic surgery, ovariectomy was performed and ovarian cortices were dissected into strips for vitrification. Some pieces were examined histologically. After warming, two to three strips were fragmented into smaller cubes before culturing with Akt stimulators for 2 days. After washing, ovarian cubes were transplanted beneath the serosa of Fallopian tubes under laparoscopic surgery. Follicle growth was monitored by ultrasound and serum estrogen levels. After oocyte retrieval from mature follicles, IVF was performed. MAIN RESULTS AND THE ROLE OF CHANCE: Among 37 patients, 54% had residual follicles based on histology. Among patients with follicles, 9 out of 20 showed follicle growth in auto-grafts with 24 oocytes retrieved from six patients. Following IVF and embryo transfer into four patients, three pregnancies were detected based on serum hCG, followed by one miscarriage and two successful deliveries. For predicting IVA success, we found that routine histological analyses of ovarian cortices and shorter duration from initial POI diagnosis to ovariectomy are valid parameters. LIMITATIONS, REASONS FOR CAUTION: Although our findings suggest that the present vitrification protocol is effective for ovarian tissue cryopreservation, we have not compared the potential of vitrification and slow freezing in follicle growth after grafting. We chose the serosa of Fallopian tubes as the auto-grating site due to its high vascularity and the ease to monitor follicle growth. Future studies are needed to evaluate the best auto-grafting sites for ovarian tissues. Also, future studies are needed to identify biological markers to indicate the presence of residual follicles in POI to predict IVA treatment outcome. WIDER IMPLICATIONS OF THE FINDINGS: In POI patients, ovarian reserve, namely the pool of residual follicles, continues to diminish with age. If one ovary is cryopreserved at an earlier stage of POI, patients could undergo additional non-invasive infertility treatments before the final decision for the IVA treatment. Furthermore, in the cases of unmarried POI patients, cryopreservation of ovarian tissues allows their fertility preservation until they desire to bear children. STUDY FUNDING/COMPETING INTERESTS: This work was supported by Grant-In-Aid for Scientific Research (Research B: 24390376, Challenging Exploratory Research: 24659722, and Innovative Areas, Mechanisms regulating gamete formation in animals: 26114510) and by research funds from the Smoking Research Foundation, and the Takeda Science Foundation. None of the authors has a conflict of interest. TRIAL REGISTRATION NUMBER: UMIN000010828.

The role of menstrual cycle phase and AMH levels in breast cancer patients whose ovarian tissue was cryopreserved for oncofertility treatment.

PURPOSE: To determine the factors that affect oocyte extraction efficiency when using the "combined procedure". In the present "combined procedure" ovarian tissue cryopreservation and oocyte extraction from an isolated ovary, later used in In Vitro Maturation (IVM), are performed concurrently. METHODS: Data were analyzed retrospectively and obtained from the clinical records of 27 young breast cancer patients referred for fertility preservation. RESULTS: The patients' mean age was 33.7 (±3.8) years, mean serum anti-Müllerian hormone (AMH) concentration was 3.5 (±2.1) ng/ml, and mean number of extracted oocytes was 8.3 (±6.1). The phase of menstruation (follicular or luteal) did not affect either the number of oocytes extracted (P = 0.99) nor oocyte survival or maturation rates. Likewise, the number of oocytes that could be extracted was not affected by the type of laparoscopic procedure (multiple-port or single-incision laparoscopy; P = 0.94) or the molecular subtype of breast cancer (either Luminal A or B; P = 0.52). Analysis revealed that the number of extracted oocytes was well-correlated with the patient's AMH serum level and age (coefficient of correlation: 0.60 and -0.48, respectively). CONCLUSION: We conclude that the outcome of the "combined procedure" primarily depends upon the patient's serum AMH level and age. Importantly, the "combined procedure" may be used during any phase of the menstrual cycle to preserve the fertility of breast cancer patients.

Analysis of late-onset ovarian insufficiency after ovarian surgery: retrospective study with 75 patients of post-surgical ovarian insufficiency.

The primary objectives of the present study are to determine the period of onset of ovarian insufficiency after surgery and to confirm potential risk factors for ovarian insufficiency after surgery for the removal of benign ovarian cysts. Data were obtained from 75 patients who underwent surgery for benign ovarian cysts prior to the onset of ovarian insufficiency. Our analysis included 835 ovarian insufficiency patients who were referred to our institution from July 2003 to July 2013. Several epidemiological parameters of ovarian insufficiency after surgery (age at operation, period of onset of ovarian insufficiency, operation procedure, and pathological diagnosis) were investigated. Of the 835 patients who had ovarian insufficiency, 75 patients (9.0%) underwent ovarian surgery before the onset of ovarian insufficiency. Of those 75 patients, 66 patients (88.0%) underwent cystectomy. For the majority of the 75 patients the surgical indication was the presence of endometriotic cysts (57 patients; 76.0%). Twelve patients (16.0%) underwent multiple surgeries (all bilateral cystectomies). The mean age of the patients at the time of surgery was 27.8±5.5 years-old, and the mean period of onset of ovarian insufficiency was 5.8±3.8 years. In patients with cystectomy, the patient's age at the time of surgery and period of onset of ovarian insufficiency was well-correlated (coefficient of correlation; hemilateral endometriotic cystectomy: -0.64, bilateral endometriotic cystectomy: -0.61, and multiple endimetriotic cystectomy: -0.40). We found that cystectomy of endometriotic cysts is the potential risk factor for ovarian insufficiency after surgery, at times, the onset of ovarian insufficiency long after cystectomy. Therefore, it is important to monitor ovarian reserve for an extended period of time after ovarian surgery. It is particularly important to monitor ovarian reserve long-term for patients who wish to conceive in the future and to suggest a variety of infertility treatments appropriate for their ovarian reserve.

Hippo signaling disruption and Akt stimulation of ovarian follicles for infertility treatment.

Primary ovarian insufficiency (POI) and polycystic ovarian syndrome are ovarian diseases causing infertility. Although there is no effective treatment for POI, therapies for polycystic ovarian syndrome include ovarian wedge resection or laser drilling to induce follicle growth. Underlying mechanisms for these disruptive procedures are unclear. Here, we explored the role of the conserved Hippo signaling pathway that serves to maintain optimal size across organs and species. We found that fragmentation of murine ovaries promoted actin polymerization and disrupted ovarian Hippo signaling, leading to increased expression of downstream growth factors, promotion of follicle growth, and the generation of mature oocytes. In addition to elucidating mechanisms underlying follicle growth elicited by ovarian damage, we further demonstrated additive follicle growth when ovarian fragmentation was combined with Akt stimulator treatments. We then extended results to treatment of infertility in POI patients via disruption of Hippo signaling by fragmenting ovaries followed by Akt stimulator treatment and autografting. We successfully promoted follicle growth, retrieved mature oocytes, and performed in vitro fertilization. Following embryo transfer, a healthy baby was delivered. The ovarian fragmentation-in vitro activation approach is not only valuable for treating infertility of POI patients but could also be useful for middle-aged infertile women, cancer patients undergoing sterilizing treatments, and other conditions of diminished ovarian reserve.

[The possibility of the fertility preservation in young cancer patients based on practice of oncofertility treatment].

Young female cancer patients face various problems, including a decrease in their quality of life(QOL)due to early menopause or loss of fertility after remission. Chemotherapy and radiotherapy can cause loss of reproductive function in young women due to adverse effects such as ovarian failure. The frequency of ovarian failure depends on the age of the patient, the anticancer agents used, and the dose of each agent. In these patients, improvement of the post-treatment QOL and fertility preservation can be achieved by measures such as protection of ovarian function against the effects of anticancer agents. Ovum freezing or fertilized egg freezing are also becoming fertility preservation methods for these patients. However, ovarian hyperstimulation to obtain ova is time consuming and sometimes considered taboo depending on the type of cancer. A self solution to problems occurring frequently at the same time is demanded from the patient, and the patient is forced to deal with too many choices in too little time. It is often less than one month until the cancer treatment begins after an underlying disease is diagnosed, since chemotherapy cannot be delayed. In these cases, cryopreservation of ovarian tissue is currently proposed for fertility preservation. In this manuscript, I will discuss a topic about fertility preservation in young cancer survivors including recent knowledge regarding cryopreservation of ovarian tissue.

Gonadotropins up-regulate the expression of enolase 2, but not enolase 1, in the rat ovary.

It has been demonstrated that the glycolytic enzymes, enolase 1 (ENO1) and enolase 2 (ENO2), are expressed in the rat ovary. In the present study, we found that mRNA levels of ovarian ENO2 but not ENO1 in normal cycling adult female rats changed significantly during the estrous cycle: ovarian ENO2 mRNA levels at metestrus were lower than those at estrus. Single injection of human CG (hCG) or equine CG (eCG) into immature (3 week old) rats up-regulated ovarian expression of ENO2. hCG mainly increased ENO2 expression in oocytes and theca cells of preantral and antral follicles, and eCG did in theca cells of these follicles. In contrast, hCG and eCG did not affect the expression of ENO1, which was mainly expressed in granulosa cells. These results suggest that endogenous gonadotropins up-regulate expression of ENO2 in oocytes and theca cells of preantral and antral follicles, which would activate glycolysis in these cells. It is also suggested that the activated glycolysis is necessary for ovarian functions such as follicle growth and maturation, and hormone production.