Preliminary results of a counselling programme for fertility preservation in female cancer patients: The experience of the GEMME DORMIENTI network.

OBJECTIVE: To describe a population of patients referred for fertility preservation (FP), how to efficiently provide FP care, and how FP care changed over time. METHODS: This longitudinal observational study enrolled 281 female cancer patients referred between 2013 and 2016 to the non-profit organisation Gemme Dormienti ONLUS (GD) for FP care. All patients underwent the same battery of instrumental and laboratory diagnostic tests. GnRHa therapy was started at least seven days before CTh treatment. RESULTS: From 2013 to 2016, we observed a progressive increase in the number of patients referred for FP care. Out of 251 eligible patients, 135 patients were treated with GnRHa only, and 72 patients underwent GnRHa therapy and cryopreservation. The median time from GD referral to oocyte and ovarian tissue cryopreservation was 11 and 5 days respectively. Tissue cryopreservation requests increased during our study period (from four cases in 2013 to 17 cases in 2016). During follow-up, 17ß-estradiol and FSH levels were significantly increased (p < .0001), and AMH levels were significantly decreased (p < .0001). CONCLUSION: The rapid increase in the number of patients who requested FP care and in the complexity of FP procedures overtime reflects the need to improve quality of life for cancer patients.

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.

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.

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.

The techniques and outcomes of hysteroscopic metroplasty.

PURPOSE OF REVIEW: To evaluate the reproductive outcomes and surgical techniques of the hysteroscopic metroplasty in women with septate uterus and recurrent abortions or primary unexplained infertility. RECENT FINDINGS: Septate uterus is the most frequent congenital uterine anomaly caused by inadequate resorption of the Müllerian ducts. Hysteroscopic metroplasty has replaced the traditional laparotomy approach because of its positive and satisfactory outcomes in pregnancy and live-birth rates, and also many different postoperative benefits. The aim of metroplasty is to restore a normal anatomy of the uterine cavity as a prerequisite for a positive implantation and subsequent good obstetrical outcomes. This treatment clearly demonstrates its effectiveness both in recurrent abortion and in primary unexplained infertility. SUMMARY: The hysteroscopic metroplasty with its simplicity, safety, and improved reproductive outcomes has liberalized the approach to treatment. Today, hysteroscopic metroplasty is a common practice to treat septate uterus with salutary effects both in infertile patients and in patients with recurrent pregnancy loss or premature labor, especially if in-vitro fertilization is being contemplated. Decisions on when to treat uterine septa are discussed in particular because of lack of prospective, randomized controlled trials.

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.

Body imaging and sexual behavior in lean women with polycystic ovary syndrome.

INTRODUCTION: In women with polycystic ovary syndrome (PCOS), changes in body appearance may influence the feminine identity of the patients with possible consequent depression and sexual dysfunction. AIM: The study aims to examine the differences in mood, perceived body image, sexual behavior, and clitoral vascularization between lean PCOS patients and healthy eumenorrheic controls. METHODS: Thirty-three lean PCOS women (Group I) and 22 healthy nonhirsute volunteers (Group II) were submitted, on day 3-5 of the cycle, to ultrasonographic (US) and Doppler analyses, to clinical, hormonal, and biochemical evaluations, and to psychometric tests. MAIN OUTCOMES MEASURES: Main outcome measures are Ferriman-Gallwey score (FG), clitoral volume, clitoral artery Pulsatility Index, the two-factor Italian McCoy female questionnaire (MFSQ), the Stunkard Figure Rating Scale (FRS), and the Beck Depression Inventory (BDI) questionnaire. RESULTS: The FG score and the androgens resulted, as expected, more elevated in PCOS patients than in controls. However, the US assessment of the clitoral body volume and the resistances registered at the level of the dorsal clitoral artery did not show any difference between Group I and Group II patients. Moreover, the two-factor Italian MFSQ, the FRS, and the BDI were similar in both groups. CONCLUSIONS: It seems that in lean PCOS women, the moderate hirsutism and hyperandrogenism do not have any important influence on body image and self-esteem and, as a consequence, on sexual function.

Ovulatory effects of flutamide in the polycystic ovary syndrome.

The long-term effects and tolerability of flutamide (Flu) on the menstrual cycle and the ovulatory function of hyperandrogenic women with or without polycystic ovary syndrome (PCOS) were investigated. The study included 118 white women with the diagnosis of various forms of hyperandrogenism (80 PCOS and 38 non-PCOS patients). Patients received Flu for three years at dosages that declined each of the three years (250, 125 and 62.5 mg/day). Sex steroid, ovulatory function and menstrual profile at baseline and during each year of Flu treatment were evaluated. Flu therapy showed a significant increase in the percentage of cycles that were ovulatory (with progesterone concentrations greater than 4 ng/ml in mid-luteal phase) and concomitant regularization of the menstrual profile in PCOS patients. A luteinizing hormone decrease and an estrone, estradiol and 17-hydroxyprogesterone increase are also observed during treatment. Regular cycles persisted in non-PCOS patients. The results were maintained during the three years of treatment. The study suggests that Flu is a satisfactory therapeutic regimen of the chronic anovulation and the irregularities of the menstrual cycle in hyperandrogenic PCOS patients in the long run.

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.

Effects of cyclic increase in gonadotropins on the in vitro development of primordial follicles to antral stage.

The aim of this study was to evaluate the effects of FSH and LH on follicle development during a long-term culture of cryopreserved human ovarian tissue, using morphological and ultrastructural examinations. Thawed ovarian tissue slices from a 4-year-old child with Wilms tumor were cultured for 32 weeks in two different culture conditions, without (medium A) and with (medium B) a monthly peaked increase in FSH and LH. At week 32, in the medium B cultured tissue, a cluster of preantral follicles associated with two oocytes prematurely ovulated was observed, suggesting that the cyclic increase of gonadotropins promoted thawed follicles to grow up to the antral stage. However, the integrity and coordinated follicle development were not maintained. Indeed, ultrastructural analysis showed a well-preserved "naked" oocyte with concomitant features of immaturity and maturity, as if this culture condition had led to an asynchronous maturation of oocyte cytoplasmic components.

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.

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.

Follicle features in adolescent and young adult women with Hodgkin's disease prior to chemotherapy: a preliminary report.

Treatment of Hodgkin's disease (HD) has improved over the last two decades, but infertility is a common side effect of chemotherapy. Men with HD have an increased risk of inadequate semen quality and severe damage of fertility before treatment. The aim of this study was to investigate the possible association between the disease itself and germ-cell damage in female patients before treatment for HD. The ovarian cortex from 13 untreated women with HD was processed by light and transmission electron microscopy and compared with the ovarian cortex from the control group of 13 untreated women without HD. Histological examination of fresh ovarian cortex showed that in HD patients, follicles presented an unusual presence of clear vacuoles when compared with control group (73.7% versus 5.7%). Ultrastructural examination demonstrated that the small vacuoles fused into large autophagic vacuoles. The percentage of vacuolated follicles was higher in patients with advanced stage and bulky disease. In conclusion, this study demonstrates for the first time that significant damage to gametes occurs in women with HD. Further investigations are required to establish whether the present features should be considered a common side effect of HD.

Prospective cohort study on the effects and tolerability of flutamide in patients with female pattern hair loss.

BACKGROUND: Flutamide has been rarely used for the treatment of female pattern hair loss (FPHL). Flutamide treatment for FPHL has not been evaluated in long-term studies with sufficiently large numbers of women. OBJECTIVE: To evaluate long-term effects, safety, and tolerability of flutamide in women with FPHL. METHODS: A prospective cohort study was conducted in our tertiary care university hospital. The cohort included 101 women diagnosed with FPHL from January 1991 to January 2006. These women received yearly reducing doses (250, 125, and 62.5 mg/day) of flutamide for 4 years. The cohort included 33 patients treated with flutamide alone and 68 treated with flutamide combined with an oral contraceptive. Clinical and endocrine evaluations were assessed semiannually and annually, respectively, in the first 3 years of the study, and once in the following year. Liver function was evaluated quarterly. RESULTS: Both groups showed a marked decrease in alopecia scores after 12 months of flutamide therapy, compared with baseline values. The maximum drug effect occurred after 2 years and was maintained during the following 2 years of treatment. Androgens were strongly suppressed. During the first year of treatment, 4% of patients abandoned the study due to hepatic disorders related to the drug. During the following years, with the lower treatment regimen, no patient abandoned the study because of hepatic alterations. CONCLUSIONS: Flutamide is a satisfactory therapeutic regimen for treatment of FPHL in the long run. Moreover, the use of very low doses (62.5 mg/day) of flutamide is associated with complete hepatic tolerability and high adherence.

Ultrasonographic extended-view technique for evaluation of abdominal fat distribution in lean women with polycystic ovary syndrome.

OBJECTIVE: To estimate whether, by using a new ultrasonographic technique (extended view; XTD view), young lean women with polycystic ovary syndrome (PCOS) have a more android fat distribution in comparison with normally menstruating women with ultrasonographic evidence of polycystic ovaries (PCO) and healthy control subjects, matched for both age and body mass index. DESIGN: Prospective observational study. SETTING: University Hospital. SAMPLE: Forty-nine lean women with PCOS, 42 eumenorrheic women with bilateral PCO and 40 healthy volunteers with regular ovulatory cycles. METHODS: Fasting blood sampling, ultrasonographic and Doppler analyses and blood pressure monitoring. MAIN OUTCOME MEASURES: Medical examination, biochemical and hormonal parameters, ultrasonographic abdominal fat measurements, ultrasonographic evaluation of carotid intima-media thickness and Doppler analysis of ophthalmic artery. An oral glucose tolerance test was performed to analyze glucose, insulin and C-peptide levels. RESULTS: The XTD ultrasonographic preperitoneal area was significantly larger in women with PCOS than in control subjects (p=0.011). The preperitoneal/subcutaneous ratio was significantly higher in women with PCOS (1.1±0.26) compared with women with PCO (0.84±0.13; p=0.05) and control women (0.67±0.13; p<0.001). The mean pulsatility index of ophthalmic arteries was higher in the PCOS women (1.93±0.57) than in control subjects (1.84±0.38; p=0.041). Total cholesterol, triglycerides and LDL cholesterol were significantly higher in women with PCOS than in those with PCO and in control subjects. CONCLUSIONS: Women with PCOS have an android fat pattern correlated with an age-dependent increased risk of cardiovascular diseases.