Altered morphokinetics in equine embryos from oocytes exposed to DEHP during IVM.

Di-(2-ethylhexyl) phthalate (DEHP) is a commonly used plasticizer with endocrine-disrupting properties. In this study, we used an equine model to investigate DEHP concentrations in ovarian follicular fluid (FF), and to determine the effects of exposure of oocytes to potentially toxic concentrations of DEHP during in vitro maturation (IVM) on embryo development and quality. Embryo development was evaluated using time-lapse monitoring (TLM), a photomicroscopic tool that reveals abnormalities in cleavage kinetics unobservable by conventional morphology assessment. Blastocyst bioenergetic/oxidative status was assessed by confocal analysis. The possibility that verbascoside (VB), a bioactive polyphenol with antioxidant activity, could counteract DEHP-induced oocyte oxidative damage, was investigated. DEHP was detected in FF and in IVM media at concentrations up to 60 nM. Culture of oocytes in the presence of 500 nM DEHP delayed second polar body extrusion, reduced duration of the second cell cycle, and increased the percentage of embryos showing abrupt multiple cleavage, compared with controls. Mitochondrial activity and intracellular levels of reactive oxygen species were reduced in blastocysts from DEHP-exposed oocytes. VB addition during IVM limited DEHP-induced blastocyst damage. In conclusion, DEHP is detectable in equine FF and culture medium, and oocyte exposure to increased concentrations of DEHP during IVM affects preimplantation embryo development. Moreover, TLM, reported for the first time in the horse in this study, is an efficient tool for identifying altered morphokinetic parameters and cleavage abnormalities associated with exposure to toxic compounds.

Transplantation of cryopreserved ovarian tissue in a patient affected by metastatic struma ovarii and endometriosis.

In this case report, the outcomes of cryopreserved ovarian tissue transplantation performed in a patient affected by struma-ovarii associated with mature cystic teratoma, recurrent endometriotic cysts and diffuse peritoneal malignant struma-ovarii implants were described. Before cryopreservation, the patient underwent two left ovarian surgeries for enucleation cysts 8 years after righ salpingo-oophorectomy for struma-ovarii. Ovarian biopsy was collected in another hospital and transported to our laboratory for cryopreservation. The patient was submitted to radioiodine-therapy for metastases from malignant struma-ovarii. After treatment she experienced premature ovarian failure. Ten years after cryopreservation, a first orthotopic transplantation was performed in the left ovary and in a peritoneal pocket. Before transplantation, ovarian samples were analyzed to assess neoplastic contamination and tissue quality. Three years later, a second transplantation was heterotopically performed in abdominal subcutaneous sites. The analysis on thawed ovarian tissue did not reveal micrometastasis and they showed follicle and stroma damages. After transplantation few small follicles were observed at ultrasound examination and hormonal levels remained at menopausal values. To date no ovarian function recovery has been observed. The report highlights that ovarian tissue cryopreservation after multiple ovarian surgery may have some limitations. An accurate counseling should be offered to patients who wish to preserve fertility.

Doxorubicin and cisplatin induce apoptosis in ovarian stromal cells obtained from cryopreserved human ovarian tissue.

AIM: To investigate mechanisms by which doxorubicin (DOX) and cisplatin (CIS) cause human ovarian stroma injury. PATIENTS & METHODS: Stromal cells from human cryopreserved ovarian tissue were cultured in the presence of 1 µM DOX and 10 µM CIS. Ovarian damage induced by treatments was evaluated by 'Live/Dead' and sulforhodamine-B assays, the expression of different apoptosis markers. RESULTS: Stromal cell growth was inhibited by DOX and CIS, and this effect was accompanied by apoptosis through mitochondrial pathway activation: Bax, cleaved-caspase 9, cleaved-PARP1 induction and Akt1, Bcl2, phospho-44/42-MAPK/ERK1/2 reduction were observed. CONCLUSION: DOX and CIS induced apoptosis in human ovarian stromal cells. Knowledge of mechanisms by which the drugs act is important to identify possible ways to counteract side effects of chemotherapy on ovaries.

New insights in the selection and management of cancer patients applicants for ovarian tissue cryopreservation.

Ovarian tissue cryopreservation (OTC), representing a promising strategy to preserve ovarian function in cancer patients, is recommended to women younger than 35 years. This study aimed to identify endocrine and biometric parameters as additional selection criteria for OTC. One hundred and ninety-one cancer patients before chemoradiotherapy and OTC and 43 controls were investigated. Mean ± SD, median, quartiles, 5th and 95th centiles and correlations of FSH, LH, estradiol, inhibin-B, anti-Mullerian hormone (AMH), ovarian volume and antral follicle count (AFC) were assessed. Most ovarian reserve parameters presented typical variations of ovulatory menstrual cycle, except AMH and AFC showing minimal fluctuations across the menstrual cycle. The 5th centiles of AMH (0.31and 0.4 ng/mL in controls and cancer patients, respectively) and AFC (five follicular structures in both groups) could be conjectured as minimum thresholds to include patients aged <35 years in OTC; below this threshold patients of any age should be excluded from OTC. Conversely, patients with AMH and AFC above the 25th centiles (1.2-1.6 ng/mL and 9-10 follicular structures in controls and cancer patients, respectively) might be inserted in OTC regardless of age. Baseline assessment of AMH and AFC might be considered as selection criteria, in addition to chronological age, to take decision of OTC in cancer patients.

Autotransplantation of cryopreserved ovarian tissue in oncological patients: recovery of ovarian function.

AIM: To present preliminary results of autotransplantation of cryopreserved ovarian tissue performed at Sant'Orsola-Malpighi Hospital, Bologna, Italy. MATERIALS & METHODS: Orthotopic transplantation was performed in two women with colorectal and breast cancer, and heterotopic transplantation was performed in one Hodgkin's lymphoma woman. The presence of micrometastasis in the ovarian tissue was checked, and morphological features of ovarian tissue were evaluated before transplantation. Ovarian function was monitored by hormonal and ultrasound-color Doppler examination after transplantation. RESULTS: In all three women, no micrometastasis was found; light and transmission electron microscopy showed well-preserved thawed ovarian tissue. Ovarian function recovery was observed 2-4 months after transplantation. Spontaneous menstrual cycles occurred in two women with normal follicular densities. No periods occurred in the woman with low follicular density at the time of tissue collection. CONCLUSION: Ovarian tissue cryopreservation and transplantation is a promising approach for preserving ovarian function in women with cancer.

Anti-Müllerian hormone as an ovarian reserve marker in young cancer women who undergo ovarian tissue cryopreservation.

AIM: To evaluate if anti-Müllerian hormone (AMH) is a reliable marker of ovarian reserve in young women undergoing ovarian tissue cryopreservation. PATIENTS & METHODS: Relationships of serum AMH levels with primordial follicle density, age and reproductive hormones were investigated using the Pearson or Spearman correlation coefficient in 86 women with cancer (12-38 years) undergoing ovarian tissue cryopreservation. AMH variations through the menstrual cycle were assessed by the Kruskal-Wallis test. p < 0.05 was accepted as significant. RESULTS: AMH positively correlated with primordial follicle density (p = 0.03), showed great interindividual variability at all ages and negatively correlated with estradiol (p = 0.007) in the early follicular phase. AMH did not vary across the menstrual cycle (p = 0.415). CONCLUSION: AMH appears a valid ovarian reserve marker in young cancer women.

Ovarian tissue biopsy for cryopreservation by vaginal natural orifice transluminal endoscopic surgery: a new approach for a minimal invasive ovarian biopsy.

OBJECTIVE: Vaginal natural orifice transluminal endoscopic surgery (vNOTES) is an emerging surgical procedure that combines the advantages of the vaginal approach with laparoscopic vision and instrumentation. Shorter hospitalization and lesser postoperative pain associated with vNOTES may be explained by the advantages of this innovative surgical approach (e.g., absence of abdominal incisions, shorter operative time, and lower insufflation pressure). Ovarian tissue cryopreservation allows to preserve reproductive and endocrine functions in young women with oncological disease at risk of premature ovarian insufficiency (POI) caused by gonadotoxic treatments. Ovarian tissue biopsy for cryopreservation consists of a large biopsy of 1 or both ovaries that is usually performed by laparoscopy. Then, the removed ovarian tissue is cryopreserved for the future transplant after cancer remission. The volume of ovarian biopsy ranges from 50% of the ovary for women at moderate risk of POI to 70%-100% of it for those at high risk. The inclusion criteria for ovarian tissue cryopreservation are women aged <35 years who cannot delay start of oncological treatments for follicle cryopreservation, with a moderate or high risk of POI and good chance of 5-year survival. Ovarian tissue cryopreservation cannot be performed if tumor treatments include uterine irradiation or for tumors at risk of ovarian metastases (as in the case of ovarian cancer, leukemia, neuroblastoma, or Burkitt lymphoma). Despite widespread adoption of vNOTES in gynecology, ovarian biopsy for cryopreservation has never been performed using this route. DESIGN: Step-by-step explanation of the procedure with descriptive text and narrated video footage. SETTING: Tertiary-level referral academic center. PATIENT(S): A 27-year-old patient recently diagnosed with low-grade follicular non-Hodgkin lymphoma was referred to our center for ovarian tissue cryopreservation before chemotherapy. The patient included in this study gave informed consent for publication of the video and posting of the video online including social media, the journal website, scientific literature websites (e.g., PubMed, ScienceDirect, and Scopus), and other applicable sites. Because of the nature of the study, institutional review board approval was not required. INTERVENTION(S): Access to the peritoneal cavity was created by a 3-cm posterior colpotomy. The peritoneum was then opened using cold scissors and temporarily fixed to the posterior vaginal wall. The GelPOINT Mini Advanced Access Platform (Applied Medical, Rancho Santa Margarita, CA), with 1 10-mm and 2 5-mm trocars, was used as the vNOTES port. The inner Alexis ring of the GelPOINT was inserted through the colpotomy into the pouch of Douglas. A hysterometer was placed into the uterine cavity to keep the uterus anteverted during the surgery. A pneumoperitoneum was created to a pressure of 8 mm Hg, and the operating table was tilted to a 20° Trendelenburg position. A 10-mm rigid 30° camera was inserted in the inferior and larger trocar, and both ovaries were visualized. Seventy percent of the left ovary was removed with cold scissors to minimize trauma on the surgical specimen. After removal of the GelPOINT cap, ovarian biopsy was immediately picked up by the biologist of our fertility center. The ovary was coagulated with a bipolar instrument. The hysterometer was then replaced by a uterine manipulator to perform tubal patency test, and blue dye passage through both salpinges was observed. Finally, the Alexis retractor and stich on the posterior peritoneum were removed, and the vagina was sutured using interrupted stiches. The total operative time was 25 minutes. MAIN OUTCOMES MEASURE(S): Ovarian tissue biopsy for cryopreservation by vNOTES. RESULT(S): No intraoperative and postoperative complications were reported, and the patient was discharged after 24 hours from surgery. CONCLUSION(S): Vaginal natural orifice transluminal endoscopic surgery may be a feasible alternative approach to laparoscopy for ovarian tissue cryopreservation: it allows an easy access to the ovaries and removal of different tissue volumes. Patients undergoing ovarian cryopreservation may benefit from the vNOTES approach because a rapid postoperative recovery is crucial to start chemotherapy in a short time. As for other vNOTES procedures, accurate selection of patients seems to be crucial for a successful ovarian tissue cryopreservation. We believe that the inclusion and exclusion criteria reported for other gynecologic procedures performed through vNOTES may also be valid for ovarian tissue cryopreservation by vNOTES. Women at high risk of pelvic adhesions (e.g., coexistent endometriosis, previous pelvic surgery, or inflammatory pelvic disease), those with an increased body mass index or enlarged uterus, and those with cervical, vaginal, or uterine cancer cannot be considered for this approach because all these factors are associated with failure of vNOTES. On the other hand, women with no history of surgery, endometriosis, and large myomas may benefit from the vNOTES approach, and these women represent most of patients who undergo ovarian tissue cryopreservation. Further and larger studies are needed to assess the efficacy and safety of this new approach.

Laparoscopic medulla-sparing ovarian tissue biopsy for cryopreservation: step-by-step surgical technique.

OBJECTIVE: To describe a laparoscopic technique for ovarian tissue biopsy (OTB) for fertility preservation. In the last years, the demand for fertility preservation has grown because of the increasing survival rates among patients with cancer and the rising awareness of the importance of quality of life after gonadotoxic therapy. Among fertility-sparing approaches, ovarian tissue cryopreservation is a valid strategy to preserve ovarian endocrine and reproductive function in prepubertal and postpubertal women who will undergo gonadotoxic cancer treatments. Currently, there is no universal consensus regarding ovarian tissue retrieval technique for fertility preservation. DESIGN: Step-by-step description of the surgical technique with narrated video footage. SETTING: Academic tertiary hospital. PATIENT(S): Patients with a high risk of premature ovarian insufficiency, usually due to gonadotoxic treatments, who undergo OTB for fertility preservation were included in the study. In this video, we present the clinical case of a 28-year-old patient affected by Hodgkin lymphoma who underwent laparoscopy for OTB before chemotherapy. INTERVENTION(S): After exposing the chosen ovary, an incision at the tubal pole of the ovary is made with scissors. Through section and dissection, a large cortical biopsy of the ovary is performed without removing and avoiding any damage to the medulla. At the end of the procedure, hemostasis was achieved with selective coagulation using bipolar coagulation. MAIN OUTCOME MEASURE(S): Step by step educational video. RESULT(S): The post-operative course was uneventful and the patient was discharge 24 hours after surgery. CONCLUSION(S): Standardization of a step-by-step laparoscopic technique can provide an effective method to optimize ovarian tissue removal while minimizing tissue injury. Medulla-sparing ovarian biopsy allows retrieval of only the cortical part of the ovary, maximizing the number of primordial follicles obtained without damaging the vascular supply of the ovary contained within the medulla. Primordial follicles are resistant to cryoinjury owing to their relatively inactive metabolism, and they are usually found at approximately 0.8 mm below the surface of the cortex. This technique could also reduce the back-table processing time of the ovarian tissue before cryopreservation. One disadvantage could be the difficulty of the technique compared to an oophorectomy because it requires a skilled surgeon that can easily find the cleavage plane between the medulla and the cortex, even in patients submitted to previous chemoradiotherapy or during gonadotropin-releasing hormone analogue therapy.

Ovarian tissue transplantation: 10 years of experience at the Bologna University.

Objective: The efficiency of ovarian tissue transplantation (OTT) was established in terms of ovarian function recovery (95% of cases), number of live births (over 200 worldwide to date) and induction of puberty. Unfortunately, the lack of international registries and the fact that many centers have not yet reported their outcomes, lead to poor knowledge of the exact fertility data. The aim of the study is to describe our experience with OTT to restore ovarian function and fertility. Methods: This study was designed as a single-center, observational, retrospective, cohort study that includes women who underwent OTT between December 2012 and June 2023 at our center. After approval by the oncologist/hematologist, a small fragment of ovarian tissue was thawed and analyzed to detect the presence of micrometastases before OTT. Thawed ovarian tissue was grafted laparoscopically at multiple sites, including the remaining ovary and pelvic side wall (orthotopic transplantation) and/or abdominal wall (heterotopic transplantation). After OTT, ovarian function was monitored by hormonal assay, ultrasound and color Doppler at approximately 4-week intervals. Results: Between December 2012 and June 2023, 30 women performed OTT. Prior to OTT, immunohistochemical and molecular analyses revealed no micrometastases in all thawed ovarian tissue samples. In our series of 30 women, 20 of women were on premature ovarian insufficiency (POI), and the remaining ten cases still had oligomenorrhea and difficulty getting pregnant. Among the women with POI before OTT and at least 6 months follow-up, recovery of endocrine function was observed in all but one woman who underwent orthotopic transplantation (13 of 14 cases), in one out of two women who underwent both orthotopic and heterotopic transplantation (1 of 2 cases) and in all women who underwent heterotopic transplantation (4 of 4 cases). Women who underwent OTT to enhance fertility had no alterations in menstrual cycle and hormonal levels. In total, ten pregnancies were obtained in 25 women, resulting in four live births, two ongoing pregnancies and four spontaneous abortions. Conclusion: Our data can help patients and physicians in their discussions and decisions about the need and possibilities of preserving fertility.

Molecular Factors Predicting Ovarian Chemotoxicity in Fertile Women: A Systematic Review.

Background: Recent advances in cancer diagnosis and treatment have significantly improved survival rates among women of reproductive age facing cancer. However, the potential iatrogenic loss of fertility caused by chemotherapeutic agents underscores the need to understand and predict chemotherapy-induced ovarian damage. This study addresses this gap by systematically reviewing the literature to investigate genetic markers associated with chemotherapy-induced ovarian failure (CIOF). Objective: The primary objective is to identify genetic markers linked to CIOF, contributing to a comprehensive understanding of the factors influencing fertility preservation in female cancer survivors. Methods: A systematic review was conducted using PubMed, EMBASE, Web of Science, Scopus, and OVID electronic databases from inception through December 2023. Studies were included if they featured genomic assessments of genes or polymorphisms related to CIOF in women with histologically confirmed tumors. Exclusion criteria comprised in vitro and animal studies, reviews, and pilot studies. The resulting four human-based studies were scrutinized for insights into genetic influences on CIOF. Results: Of the 5179 articles initially identified, four studies met the inclusion criteria, focusing on alkylating agents, particularly cyclophosphamide, and anthracyclines. Su et al. explored CYP3A41B variants, revealing modified associations with CIOF based on age. Charo et al. investigated GSTA1 and CYP2C19 polymorphisms, emphasizing the need to consider age and tamoxifen therapy in assessing associations. Oktay et al. delved into the impact of BRCA mutations on anti-Müllerian hormone (AMH) levels post-chemotherapy, supported by in vitro assays. Van der Perk et al. focused on childhood cancer survivors and revealed significant associations of CYP3A43 and CYP2B6*2 SNPs with AMH levels. Conclusions: This systematic review analyzes evidence regarding genetic markers influencing CIOF, emphasizing the complex interplay of age, specific genetic variants, and chemotherapy regimens. The findings underscore the need for a personalized approach in assessing CIOF risk, integrating genetic markers with traditional ovarian reserve testing. The implications of this study extend to potential advancements in fertility preservation strategies, offering clinicians a comprehensive baseline assessment for tailored interventions based on each patient's unique genetic profile. Further research is essential to validate these findings and establish a robust framework for integrating genetic markers into clinical practice.

Detection of Non-Hodgkin Lymphoma Within Ovarian Tissue.

This case report describes the detection of non-Hodgkin lymphoma (NHL) within ovarian tissue after cryopreservation. The 27-year-old woman presented no gynecological symptoms such as pelvic pain or abnormal uterine bleeding. During laparoscopy for ovarian tissue cryopreservation, the ovaries appeared markedly modified and some solid content cysts were highlighted. Microscopically the cysts revealed the presence of lymphoid infiltrate, whereas the histological assessment of some fragments of the ovarian cortical tissue revealed no evidence of pathological lymphoid infiltration. This case report describes the presence of pathological lymphoid infiltration in ovarian cysts but not in the cortical tissue of a young woman with secondary NHL. Despite the absence of the positivity in cortical tissue it was recommended to avoid the cryopreserved ovarian tissue transplantation to reduce the risk of reseeding of the malignancy in the woman.

Cryopreservation of ovarian tissue in breast cancer patients: 10 years of experience.

AIM: To present a decade of experience with ovarian tissue cryopreservation in breast cancer patients. MATERIALS & METHODS: The safety of the procedure was histologically evaluated before and after freezing in 94 patients. Out of 94 patients, 48 prechemotherapy patients were randomly selected to determine stroma and follicle preservation and follicular density. RESULTS: The ovarian tissue from 94 patients did not identify any micrometastases. After cryopreservation, morphology of the ovarian tissue and density of healthy follicles were similar in fresh and frozen tissue. Follicular density decreased with the increasing age of patients in both fresh and frozen tissue (p < 0.0001). A variation in follicular density was observed between fresh and frozen tissue (p < 0.05). CONCLUSION: These results suggest that ovarian tissue cryopreservation is highly feasible for preserving the fertility of young breast cancer patients.

Impact of the COVID-19 pandemic on ovarian tissue cryopreservation planning in the Bologna clinical center.

Introduction: Treatment of patients with COVID-19 has been a priority by competing with the treatment of any other disease due to limited hospital resources. The current pandemic situation has focused the attention of healthcare providers around the world away from all other non-emergency health problems, including oncofertility. The aim of the study was to evaluate the repercussions of the COVID-19 pandemic on the activity levels of ovarian tissue cryopreservation (OTC) in the our center. Methods: The study analyzed the number of patients treated for OTC in our center during three periods: pre-pandemic period: March 2019-February 2020, pandemic period: March 2020-February 2021 and post-pandemic period: March 2021-February 2022. Results: In our center routine hospital operation was completely reorganized, allowing only urgent interventions. Continuing to urgently preserve fertility during the pandemic required rapid changes to our standard practices for the care of these vulnerable patients. Despite the modifications, there was no difference in the number of OTC performed among the periods analyzed. Similarly, the number of patients who did not perform OTC was the same over the three years analyzed. Discussion: Despite the local and national restructuring of care to conserve resources and protect the community, it is significant to continue offering fertility-sparing treatment to cancer patients. This emphasis on the importance of preserving fertility despite the pandemic further highlights the essential and urgent nature of this procedure.

Cost-effectiveness analysis of ovarian tissue cryopreservation and transplantation for preservation of fertility in post-pubertal oncological women submitted to high-risk gonadotoxic chemotherapy.

OBJECTIVE: To study the economic impact of ovarian tissue cryopreservation and transplantation (OTC) in post-pubertal patients who underwent high-risk gonadotoxic chemotherapy. METHODS: A decision tree model was used to determine the live birth rate and cost-effectiveness of OTC versus non-OTC. The incremental cost-effectiveness ratio (ICER) was calculated. A sensitivity analysis was performed under the assumption that the costs of ovarian cortex retrieval, cryopreservation, and storage for patients with cancer might be covered by the national health system or health insurance. RESULTS: Patients had the greatest probability of achieving live birth after high-risk chemotherapy when they underwent OTC versus non-OTC. Although cryopreservation of ovarian tissue results in higher live birth rates, it is always more expensive. Cost-effectiveness increases when the majority of patients completes the path of tissue cryopreservation plus transplantation after 5 years. CONCLUSION: Although OCT has been demonstrated as a procedure for effective fertility preservation in fertility-age women with cancer, no cost-effectiveness analysis has been performed until now. This model could help healthcare systems to allocate coverage for OCT.

Corrigendum: Ovarian tissue cryopreservation and transplantation: 20 years experience in Bologna University.

[This corrects the article DOI: 10.3389/fendo.2022.1035109.].

Ovarian tissue cryopreservation and transplantation: 20 years experience in Bologna University.

Objective: To report the 20-year experience in ovarian tissue cryopreservation (OTC) and ovarian tissue transplantation (OTT) of the Bologna clinical center (Bologna, Italy). Design: Retrospective cohort study. Patients: 1026 pediatrics and women aged between 2 and 38 years who underwent OTC and OTT between January 2002 to January 2022. Results: Of the 1026 patients, 238 (22.8%) were pediatrics (≤ 17 years, Group 1) and 788 (77.2%) were adult women (range 18-38 years, Group 2). In Group 1, 184 (77.3%) patients had malignant diseases and 54 (22.7%) had non-malignant diseases. In Group 2, 746 (94.7%) patients had malignant diseases and 42 (5.3%) had non-malignant diseases. No real complications were observed during surgery. In all the samples analyzed most of the follicles were in the resting stage, while only a few follicles were growing. In both fresh and thawed samples, follicular density was higher in Group 1 than in Group 2 (p < 0.01). Regardless of age, good preservation of follicles and stroma was observed in fresh and thawed ovarian tissue by histological and immunohistochemical analyses (estrogen and progesterone receptors; Ki67 and Bcl2 markers; TUNEL). To date, out of 1026 total women, 812 (79.1%) had their tissue stored. Sixty-eight (6.6%) patients died from their primary disease. Twenty-four (2.3%) women performed 33 OTTs between December 2011 and January 2022. Restoration of menstruation was observed in 15 out of 17 menopausal women. Six pregnancies were achieved, two hesitated in abortion and four in the birth of healthy babies. Conclusion: OTC is the only fertility preservation technique applicable in pre-pubertal/pediatrics and in adult patients when stimulation for oocytes/embryos cryopreservation is not possible. The reported data can help future patients and physicians in their discussions and decisions about the need and possibilities of preserving ovarian function.

Description of the Follicular Fluid Cytokine and Hormone Profiles in Human Physiological Natural Cycles.

PURPOSE: Exogenous gonadotrophins administration during in vitro fertilization/intracytoplasmic sperm injection (IVF/ICSI) cycles could significantly alter the endogenous follicular regulation system and could influence oocyte quality. The analysis of the follicular fluid (FF) cytokine and hormone profiles in physiological natural cycles is crucial to appreciate the role of FF milieu on follicle development. So far, the FF cytokine profile has been analyzed only in controlled ovarian stimulation cycles and in modified natural cycles. Our study defines, in physiological natural cycles, the cytokine and hormone profiles of individual FF aspirated from antral follicles. METHODS: A total of 203 FFs obtained from 83 women with regular menstrual cycles undergoing ovarian tissue cryopreservation were analyzed: 115 FFs from Group 1 (10 to 29 years of age) and 88 FFs from Group 2 (30 to 40 years of age). In individual FF, 27 cytokines were measured with xMAP technology, and progesterone, estrone, estradiol, testosterone, androstenedione concentrations were determined by liquid chromatography-tandem mass spectrometry. RESULTS: FF hormone profiles were not different in follicular and luteal phase, suggesting that FF hormones are regulated independently of the endogenous gonadotrophins-possibly because 74% of the punctured follicles, which were ≤6 mm, did not require cyclic pituitary function. The follicle size was influenced not only by the FF cytokine profile but also by the FF hormone profile, both of which are dependent on age. MAIN CONCLUSIONS: In physiological natural cycles, FF hormones seems to be regulated independently of the endogenous gonadotropins. Age influences FF hormone and cytokine profiles and the compelling relationship between FF hormones and FF cytokines could influence the follicle development.

Effects of low-dose X-ray medical diagnostics on female gonads: Insights from large animal oocytes and human ovaries as complementary models.

Diagnostic imaging has significantly grown over the last thirty years as indispensable support for diagnostic, prognostic, therapeutic and monitoring procedures of human diseases. This study explored the effects of low-dose X-ray medical diagnostics exposure on female fertility. To aim this, cumulus-oocyte complexes (COCs) recovered from the ovaries of juvenile sheep and human ovaries were used as complementary models for in vitro studies. In the sheep model, the effects of low-dose X-rays on oocyte viability and developmental competence were evaluated. In human ovaries originated from two age group (21-25 and 33-36 years old) subjects with gender dysphoria, X-rays effects on tissue morphology, follicular density and expression of apoptosis-related (NOXA, PUMA, Bcl2, Bak, γH2AX) and cell cycle-related genes (p21 and ki67) were investigated. It was noted that in sheep, the minimum dose of 10 mGy did not influence most of examined parameters at oocyte and embryo levels, whereas 50 and 100 mGy X-ray exposure reduced oocyte bioenergetic/oxidative activity but without any visible effects on oocyte and embryo development. In addition, blastocyst bioenergetic/oxidative status was reduced with all used doses. Overall data on human ovaries showed that low-dose X-rays, similarly as in sheep, did not alter any of examined parameters. However, in women belonging to the 33-36 year group, significantly reduced follicular density was observed after exposure to 50 and 100 mGy, and increased NOXA and Bax expression after exposure at 50 mGy. In conclusion, used low-doses of X-ray exposure, which resemble doses used in medical diagnostics, produce weak damaging effects on female fertility with increased susceptibility in advanced age.

Update on oogenesis in vitro.

INTRODUCTION: Ovarian tissue is increasingly being collected from cancer patients and cryopreserved for fertility preservation. Alternately to the autologous transplantation, the development of culture systems that support oocyte development from the primordial follicle stage represent a valid strategy to restore fertility. The aim of this study is to review the most recent data regarding oogenesis in vitro and to provide an up-to-date on the contemporary knowledge of follicle growth and development in vitro. EVIDENCE ACQUISITION: A comprehensive systematic MEDLINE search was performed since February 2018 for English-language reports by using the following terms: "ovary," "animal and human follicle," "in vitro growth and development," "ovarian tissue culture," "fertility preservation," "IVM," "oocyte." Previous published reviews and recent published original articles were preferred in order to meet our study scope. EVIDENCE SYNTHESIS: Over time, many studies have been conducted with the aim to optimize the characteristics of ovarian tissue culture systems and to better support the three main phases: 1) activation of primordial follicles; 2) isolation and culture of growing preantral follicles; 3) removal from the follicle environment and maturation of oocyte cumulus complexes. While complete oocyte in vitro development has been achieved in mouse, with the production of live offspring, the goal of obtaining oocytes of sufficient quality to support embryo development has not been completely reached into higher mammals despite decades of effort. CONCLUSIONS: Over the years, many improvements have been made on ovarian tissue cultures with the future purpose that patients will be provided with a greater number of developmentally competent oocytes for fertility preservation.

First Italian birth after cryopreserved ovarian tissue transplantation in a patient affected by non-Hodgkin's lymphoma.

This case report describes the first Italian live birth obtained by cryopreserved ovarian tissue transplantation in a woman affected by non-Hodgkin's lymphoma. Before anticancer treatments, several fertility preservation options were proposed. At 29 years the patient underwent laparoscopy for ovarian tissue cryopreservation. After treatments she experienced premature ovarian failure (POF) and asked for cryopreserved ovarian tissue transplantation. Before transplantation, ovarian samples were analyzed to assess neoplastic contamination and tissue quality. Two subsequent ovarian tissue transplantations were performed 4 and 7 years after cryopreservation. The follicle-stimulating hormone and luteinizing hormone reduction, estradiol increase and first menstrual cycle appeared 2 months after the second transplantation. The woman conceived spontaneously 5 months after the second transplantation. After 39 weeks of uneventful gestation, a healthy male baby was born. Ovarian tissue cryopreservation, thawing and transplantation successfully restored ovarian function and fertility after tissue storage.