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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.

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.

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.

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, 17β-estradiol and FSH levels were significantly increased (p

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.

Chemotherapy protocol can destroy the reproductive potential of young cancer patients. Doxorubicin (DOX) is a potent anthracycline commonly used in the treatment of numerous malignancies. The purpose of the study was to evaluate the ovarian toxicity of DOX via inflammation and the possible protective effect of the green tea polyphenol epigallocatechin-3-gallate (EGCG). Ovarian tissue of three patients was cultured with 1 µg/ml DOX and/or 10 µg/ml EGCG for 24 and 48 h. Levels of inflammatory factors were determined by quantitative Real-Time PCR, western blot, zimography, and multiplex bead-based immunoassay. Morphological evaluation, damaged follicle count and TUNEL assay were also performed. DOX influenced inflammatory responses by inducing a significant increase in the expression of pro-inflammatory cytokines, such as tumor necrosis factor-α (TNF-α) and cyclooxigenase-2 (COX-2), of inflammatory interleukins (IL), such as interleukin-6 (IL-6) and interleukin-8 (IL-8), and the inflammatory proteins mediators metalloproteinase-2 and metalloproteinase-9 (MMP2 and MMP9). IL-8 secretion in the culture supernatants and MMP9 activity also significantly raised after DOX treatment. Moreover, a histological evaluation of the ovarian tissue showed morphological damage to follicles and stroma after DOX exposure. EGCG significantly reduced DOX-induced inflammatory responses and improved the preservation of follicles. DOX-induced inflammation could be responsible for the ovarian function impairment of chemotherapy. EGCG could have a protective role in reducing DOX-mediated inflammatory responses in human ovarian tissue.

In this case report, we describe the outcomes of two heterotopic transplantations of cryopreserved ovarian tissue performed in a patient with HL, after 11 and 15 years of storage. At the age of 30, the patient underwent laparoscopy to collect ovarian tissue for cryopreservation before chemotherapy and radiotherapy. Eleven years later she experienced premature ovarian failure (POF). As the patient was only interested in endocrine function recovery, two heterotopic ovarian tissue transplantations were performed in the abdominal wall above the rectus muscle respectively 11 and 15 years after cryopreservation. Before transplantation, ovarian samples were analyzed to assess neoplastic contamination and tissue quality. The analysis on thawed ovarian tissue did not reveal micrometastasis and it showed well-preserved follicles and stroma. After both ovarian tissue grafting, menopausal symptoms ceased. The patient had periods approximately every 30-days and hormonal levels were within the premenopausal range. The endocrine function lasted 3-years after the first heterotopic transplantation and is still ongoing after second transplantation. Cryopreservation of ovarian tissue should be proposed to HL patients, as the incidence of POF as a long-term complication is not negligible. In these patients heterotopic transplantation is a useful tool to eliminate menopausal symptoms, preventing osteoporosis and reducing cardiovascular risks.

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.

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.

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.

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.

ABSTRACT: 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

ABSTRACT: 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 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.

Objective To investigate the ovarian reserve in female lymphoma patients and the potential relationships with the cytokine network. Design Age-matched control study. Setting Women's university hospital. Patient(s) Seventy-three lymphoma patients (57 with classic Hodgkin lymphoma [HL] and 16 with non-Hodgkin lymphoma [NHL]), approaching our center for ovarian tissue cryopreservation (study group) were compared with 25 age-matched healthy volunteers (control group). Intervention(s) Measurements of antimullerian hormone (AMH), soluble interleukin-2 receptor (SIL-2R), interleukin-6 (IL-6), interleukin-8 (IL-8) and tumor necrosis factor α (TNF-α) levels. Main Outcome Measure(s) The AMH and cytokine levels of the lymphoma patients and the healthy volunteers were compared. Correlations between AMH with SIL-2R, IL-6, and IL-8 levels were performed. Result(s) The AMH showed significant lower concentrations in lymphoma patients than in the control group. Higher significant concentrations in lymphoma patients than in control group were found for SIL-2R and IL-6. No differences were observed comparing HL and NHL groups and within the stages of HL group for AMH and all the cytokines analyzed. Finally, significant inverse correlations were observed in lymphoma patients between AMH and SIL-2R, IL-6, and IL-8 levels, but not with TNF-α levels. Positive correlations between SIL-2R with IL-6, and IL-6 with IL-8 were also shown. Conclusion(s) In patients with HL or NHL at baseline the cytokine network is particularly active and the ovarian reserve is reduced. A strong negative correlation between AMH and SIL-2R, IL-6, and IL-8 has been also evidenced.

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.

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

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

Background Flutamide has been rarely used (or 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.

As the number of young girls subjected to chemo and radiotherapy is steadily increasing it is our duty to try to preserve their future fertility and ovarian function. A possibility is to collect ovarian samples, cryopreserve them and reimplant the tissue when the treatments are over. We report our 11 year experience with the use of laparoscopy to collect ovarian tissue samples. In particular we focus on the details of the surgical technique. The procedure was performed in 54 patients affected by malignancies or pathologies requiring chemo or radiotherapy, with good results. Cryopreservation seems to be a good option to preserve future fertility although it is still an experimental method.

Introduction Vaginal dryness, low sexual desire, and low sexual arousability are critical issues for many postmenopausal women. Hormone therapy seems to improve vaginal lubrication and dyspareunia. Aim To evaluate, in postmenopausal women who refused hormonal therapy, the role of genistein in the treatment of vasomotor symptoms and its capacity to induce clitoral volumetric and vascular modifications independently from sexual stimulation. Methods Twenty‐nine postmenopausal women who refused hormonal therapy were submitted to oral daily treatment with genistein 45 mg (Group I; N = 15); or no treatment (Group II; N = 14). The Group II patients served as controls. The patients were not randomly assigned to the two groups. The patients were studied before and after 3 months. Main Outcomes Measures Endometrial and clitoral ultrasonographic analysis; color Doppler evaluation of the dorsal clitoral artery; evaluation of hormonal plasma concentrations; administration of the two‐factor Italian McCoy Female Sexuality Questionnaire; compilation of a monthly diary reporting the number of hot flashes. The ultrasound, color Doppler and psychometric tests were performed by examiners blinded to the participant's group assignment. Results In the genistein‐treated patients the vasomotor symptoms ameliorated at the end of the study. The use of genistein did not influence any other parameter. Conclusions Postmenopausal women submitted to a 3‐months treatment with genistein showed an improvement of vasomotor symptoms. However, isoflavones seem to not induce any modification in the clitoral structures. Battaglia C, Cianciosi A, Mancini F, Fabbri R, Busacchi P, Nappi RE, and Venturoli S. Genistein supplements might not induce clitoral modifications in postmenopausal women: A prospective, pilot study.

This paper reviews the most relevant literature from the past 10 years on different techniques for the culture of fresh and cryopreserved ovarian tissue from animals and humans. Information on strategies for culturing whole ovarian tissue and isolated follicles are provided as well as an updated and comprehensive view of the role that growth factors have in mediating and regulating in vitro folliculogenesis.

The endeavor to cryopreserve gonadal tissues and gametes in young patients who must undergo severe systemic anti-cancer treatment is entirely focused on the possibility of preserving their fertility potential. In this Review we deal with the development of ovarian tissue cryopreservation and the efforts to optimise the number of variables that appeared to affect frozen tissue survival, particularly the type and the concentration of the cryoprotectants that can best preserve the tissue integrity. We will discuss firstly the need to avoid both freezing and osmotic injury in cryopreserved tissues, secondly the question as to whether it is more useful to preserve follicles or whole ovarian tissue, and finally experiences in the animal model and in human tissues. The clinical application of these procedures are the ultimate focus of the whole discussion: what to do once a patient’s disease is cured and a patient considers reproduction? The clinicians considerations as to whether an auto-graft to the patient or engraft to immunologically nude animals, or indeed an attempt to develop an oocyte to maturity in vitro? Currently, only autografting techniques are able to lead to the resumption of both follicular growth and endogenous hormone production and even to a small number of pregnancies, either spontaneous or IVF-induced. Finally, we review our past experiences of ovarian tissue cryopreservation, from the choice of propanediol as the best cryoprotectant to the choice of the protein support which best assists tissue cryopreservation, and the recent experiences on long-term cultures of follicles from primordial to antral stage.

Study question Which is the best method for human ovarian tissue cryopreservation: slow freezing/rapid thawing (SF/RT) or vitrification/warming (V/W)? Summary answer The conventional SF/RT protocol used in this study seems to better preserve the morpho-functional status of human cryopreserved ovarian tissue than the used open carrier V/W protocol. What is known already Cryopreservation of human ovarian tissue is generally performed using the SF/RT method. However, reduction in the follicular pool and stroma damage are often observed. An emerging alternative procedure is represented by V/W which seems to allow the maintenance of the morphological integrity of the stroma. Study design, size, duration This is a retrospective cohort study including six patients affected by oncological diseases and enrolled from January to December 2014. Participants/materials, setting, methods Ovarian tissue was laparoscopically harvested from the right and left ovaries and was cryopreserved using a routinary SF/RT protocol or a V/W method, involving tissue incubation in two solutions (containing propylene glycol, ethylene glycol and sucrose at different concentrations) and vitrification in an open system. For each patient, three pieces from each ovary were collected at the time of laparoscopy (fresh tissue) and after storage (SF/RT or V/W) and processed for light microscopy (LM) and transmission electron microscopy (TEM), to assess the morphological and ultrastructural features of follicles and stroma, and for laser scanning confocal microscopy (LSCM), to determine the functional energetic/redox stroma status. The preservation status of SF/RT and V/W ovarian tissues was compared with that of fresh ones, as well as between them. Main results and the role of chance By LM and TEM, SF/RT and V/W samples showed cryodamage of small entity. Interstitial oedema and increased stromal cell vacuolization and chromatin clumping were observed in SF/RT samples; in contrast, V/W samples showed oocyte nuclei with slightly thickened chromatin and irregular shapes. The functional imaging analysis by LSCM revealed that the mitochondrial activity and intracellular reactive oxygen species levels were reduced both in SF/RT and in V/W samples compared with fresh samples. The study also showed progressive dysfunction of the mitochondrial activity going from the outer to the inner serial section of the ovarian cortex. The reduction of mitochondrial activity of V/W samples compared with fresh samples was significantly higher in the inner section than in the outer section. Limitations, reasons for caution The results report the bioenergetic and oxidative status assessment of fresh and cryopreserved human ovarian tissue by LSCM, a technique recently applied to tissue samples. The use of LSCM on human ovarian tissues after SF/RT or V/W is a new application that requires validation. The procedures for mitochondrial staining with functional probes and fixing are not yet standardized. Xenografting of the cryopreserved ovarian tissue in severe combined immunodeficient mice and in vitro culture have not yet been performed. Wider implications of the findings The identification of a cryopreservation method able to maintain the morpho-functional integrity of the ovarian tissue and a number of follicles comparable with those observed in fresh tissue might optimize results in clinical practice, in terms of recovery, duration of ovarian function and increased delivery outcomes after replanting. The SF/RT protocol allowed better morpho-functional tissue integrity than the V/W procedure. Study funding/competing interests Funding was provided by Fondazione del Monte di Bologna e Ravenna, Italy. Dr N.A.M. was granted by the project ONEV MIUR PONa3 00134-n.254/R&C 18 5 2011 and the project GR-2011-02351396 (Ministry of Health, Young Researchers Grant 2011/2012). There are no competing interests. Trial registration number Clinical trial 74/2001/0 (approved:13 2 2002): 'Pilot study on cryopreservation of human ovarian tissue: morphological and immunohistochemical analysis before and after cryopreservation'.

Oocyte cryopreservation has the potential to be an important adjunct to assisted reproductive technologies and bypasses some ethical, moral, and religious dilemmas posed by human embryo cryopreservation. The success of human oocyte cryopreservation depends on morphological and biophysical factors that could influence oocyte survival after thawing. Among the morphological factors, the maturity, quality, size of the oocyte, the presence or the absence of the cumulus oophorus seems to play an important role in oocyte survival after thawing. The main biophysical factor of cellular disruption during cryopreservation process in the intracellular ice formation that can be avoided by an adequate cell dehydration; thus reducing the intracellular water by increasing the dehydration process we can limit the damages of the cryopreservation procedure. The dehydration process can be affected by the presence and concentration of the cryoprotectants in the freezing solutions (equilibration and loading solutions), and by the freezing and thawing rate. Two additional properties of cryoprotectants help to protect cells during slow cooling, when the cells are very dehydrated and are surrounded by concentrated salts. The cryoprotectants appear to reduce damage caused by high levels of salt, a property known as salt buffering. Some events occurring to the oocyte during cryopreservation procedure has been found to be a premature exocitosis of cortical granules, leading to an intempestive zona hardening and consequently to a reduction of fertilization rate, and the cryoinjury to the zona pellucida leading to a polispermic fertilization. ICSI is an efficient method to by pass these two events and to achieve a satisfactory outcome in terms of normal fertilization of cryopreserved oocytes. The application of the ICSI to cryopreserved oocytes did not seem to increase the degeneration rate after insemination with respect to fresh oocytes. The increased oocyte survival rate and the use of ICSI have facilitated the recent increase in the number of pregnancies and live birth.

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.

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.

The aim of this study was to develop a vitrification procedure for human ovarian tissue cryopreservation in order to better preserve the ovarian tissue. Large size samples of ovarian tissue retrieved from 15 female-to-male transgender subjects (18–38 years) were vitrified using two solutions (containing propylene glycol, ethylene glycol, and sucrose at different concentrations) in an open system. Light microscopy, transmission electron microscopy, and TUNEL assay were applied to evaluate the efficiency of the vitrification protocol. After vitrification/warming, light microscopy showed oocyte nucleus with slightly thickened chromatin and irregular shape, while granulosa and stromal cells appeared well preserved. Transmission electron microscopy showed oocytes with slightly irregular nuclear shape and finely dispersed chromatin. Clear vacuoles and alterations in cellular organelles were seen in the oocyte cytoplasm. Stromal cells had a moderately dispersed chromatin and homogeneous cytoplasm with slight vacuolization. TUNEL assay revealed the lack of apoptosis induction by vitrification in all ovarian cell types. In conclusion after vitrification/warming the stromal compartment maintained morphological and ultrastructural features similar to fresh tissue, while the oocyte cytoplasm was slightly damaged. Although these data are encouraging, further studies are necessary and essential to optimize vitrification procedure.

Segregation of mutant mtDNA in human tissues and through the germline is debated, with no consensus about the nature and size of the bottleneck hypothesized to explain rapid generational shifts in mutant loads. We investigated two maternal lineages with an apparently different inheritance pattern of the same pathogenic mtDNA 3243A>G/tRNALeu(UUR) (MELAS) mutation. We collected blood cells, muscle biopsies, urinary epithelium and hair follicles from 20 individuals, as well as oocytes and an ovarian biopsy from one female mutation carrier, all belonging to the two maternal lineages to assess mutant mtDNA load, and calculated the theoretical germline bottleneck size (number of segregating units). We also evaluated "mother-to- offspring" segregations from the literature, for which heteroplasmy assessment was available in at least three siblings besides the proband. Our results showed that mutation load was prevalent in skeletal muscle and urinary epithelium, whereas in blood cells there was an inverse correlation with age, as previously reported. The histoenzymatic staining of the ovarian biopsy failed to show any cytochrome-c-oxidase defective oocyte. Analysis of four oocytes and one offspring from the same unaffected mother of the first family showed intermediate heteroplasmic mutant loads (10% to 75%), whereas very skewed loads of mutant mtDNA (0% or 81%) were detected in five offspring of another unaffected mother from the second family. Bottleneck size was 89 segregating units for the first mother and 84 for the second. This was remarkably close to 88, the number of "segregating units" in the "mother-to- offspring" segregations retrieved from literature. In conclusion, a wide range of mutant loads may be found in offspring tissues and oocytes, resulting from a similar theoretical bottleneck size.

Summary The efficacy of recombinant human follicle-stimulating hormone (rhFSH) in the treatment of normogonadotropic patients with male-factor infertility was assessed. Forty-five infertile men with moderate/severe oligoasthenozoospermia and normal FSH, luteinizing hormone (LH) and testosterone (T) levels were treated with high rhFSH dose (300 IU) on alternate days for ≥4 months. In all, the seminal parameters, endocrine profile (FSH, LH, prolactin (PRL), total and free T and estradiol) and pregnancy rate were evaluated before, during and after rhFSH treatment. Fifteen infertile men were treated with placebo and studied in the same way, as control group. rhFSH treatment induced a marked increase in sperm count and no change in sperm motility, morphology and viability. No changes in seminal parameters were observed in the placebo group. FSH levels increased during treatment with rhFSH and not with placebo. No variations in LH, PRL, free and total T and estradiol were evidenced during treatment. A significant pregnancy rate in rhFSH versus placebo patients was also highlighted. Prolonged treatment with high rhFSH doses leads to increase sperm count and improve the spontaneous pregnancy rate in normogonadotropic infertile patients with oligoasthenozoospermia. rhFSH may represent a rational and useful tool in the treatment of male-factor infertility.

Since the successful development in the mouse, the oocyte cryopreservation has been applied with varying success to a number of different species including the human. The recently reported successes in terms of pregnancies obtained by human oocyte cryopreservation are encouraging. Several studies typically reported different rates of survival (20-80%), fertilization (30-60%) and cleavage (32-100%). This variability of results throws some doubts on the usefulness of oocyte cryopreservation in IVF treatment cycles. It remains to be determined whether the relatively different success rates reported in literature, mainly in terms of survival rate, are due to methodological differences. We tried to investigate the effect of some factors on the oocyte survival rate after thawing: the presence or absence of cumulus oophorus and the exposure time of the oocytes to cryoprotectant. We suggest that a combination of several factors including both morphological and biophisical ones can affect the oocyte survival rate.

Oocyte cryopreservation is a viable solution for the ethical problems related to embryo storage, and the only available technique for preservation of fertility in women who have to undergo chemo- or radiotherapy. The main problems with oocyte cryopreservation are concerned with the survival rate and the fertilization rate. Recently the introduction of the intracytoplasmic sperm injection (ICSI) led to an increase in the fertilization rate. The success achieved with the first case treated encouraged us to set up a clinical trial on human oocyte cryopreservation. In the first stage of the study, 23 women with tubal infertility were enrolled. Superovulation was induced and 375 oocytes were retrieved; of these 338 oocytes were frozen. The survival rate was 59.5% and was independant of the duration of cryopreservation or the presence of cumulus. The normal fertilization rate was 64.4%, and only 7.5% of fertilizations were abnormal. A total of 90.8% of fertilized oocytes cleaved. A mean of 3.1±1.3 embryos per patient were transferred. Three pregnancies were achieved. In the second stage of our investigation, more patients were enrolled and similar results were observed. Sixteen pregnancies were achieved. A further stage of the investigation involved the fertilization of frozen oocytes with frozen sperm and even these resulted in a pregnancy. Our study demonstrated that pregnancies can also be achieved when frozen eggs are fertilized by testicular and epididymal sperm. As a consequence of the success of our investigations, a program of oocyte cryopreservation for oncological patients has been initiated in our centre. In our opinion, oocyte cryopreservation is, at present, a safe and efficient technique as documented by the birth of several healthy children.

The possibility to employ cryopreservation in Preimplantation Genetic Diagnosis (PGD) should enlarge the opportunities for research and clinical activity. For these purposes, we tried three kinds of approaches on human abnormal embryos: (1) cryopreservation of biopsied embryos; (2) biopsy of thawed embryos; and (3) biopsy of embryos derived from thawed oocytes. Our preliminary results show that: (1) biopsy of thawed embryos is feasible and FISH analysis is possible on both survived and lysed cells; (2) Optimization of freezing/thawing procedures are necessary to obtain better survival rate after thawing of biopsied embryos; (3) Biopsy and FISH are feasible on embryos derived from thawed oocytes and they could be a good way to study the chromosomal arrangement of these poorly investigated embryos.

Purpose: Our purpose was to determine the effects of thecoculture of embryos on human granulosa cells (GCs) inpatients in the first cycle of IVF-ET treatment and in patientswith repeated implantation failures and to investigate thepresence of specific proteins in a 48-hr GC conditionedmedium and the GC ultrastructural characteristics. Methods: Eighteen patients with tubal or idiopathicinfertility were enrolled in this study: 7 patients (Trial 1) were inthe first cycle of IVF-ET treatment and 11 patients (Trial 2)had repeated implantation failures (one to five). Embryosfrom each patient were cocultured randomly either onhomologous granulosa cells or on a conventional culturemedium. Results: At the end of the coculture period (day 5 or 6),50% of the embryos (Trial 1) reached the blastocyst stage,with respect to 35% in Trial 2. The pregnancy rate perretrieval was 14.2 and 9%, respectively, in Trial 1 and inTrial 2. Many conditioned media showed proteins of 24–29kDa. and some of them showed additional proteins of 90kDa. The ultrastructural analysis of GCs showed healthy,metabolically active, protein-synthesizing, and mostlysteroidogenic cells. Conclusions: GC cultures improve embryo development butnot pregnancy rates both in Trial 1 and in Trial 2.

Objective: To evaluate the influence of human granulosa cell (GC) cultures and follicular fluid (FF) on sperm kinetic parameters, hyperactivation, and the acrosome reaction compared with the influence of human tubal fluid (HTF) and Ham's F-10 medium. Design: Sperm kinetic parameters, hyperactivation, and the acrosome reaction were evaluated after 6 hours of incubation in HTF, Ham's F-10 medium, FF, and GC cultures. Setting: Infertility and In Vitro Fertilization Centre, Reproductive Endocrinology Unit, Institute of Obstetrics and Gynaecology. Patient(s): Sixteen normal semen samples. Intervention(s): Sperm kinetic parameters and hyperactivation were analyzed using an automated videomicrography system, the acrosome reaction was performed using a triple-stain technique, and progesterone and 17OH-progesterone levels were measured with the use of commercially available kits. Main Outcome Measure(s): Sperm kinetic parameters, hyperactivation, acrosome reaction. Result(s): The percentage of motile sperm, the mean curvilinear velocity, and the mean of the maximum amplitude of lateral head movement were increased significantly after 6 hours of incubation in FF or GC cultures compared with incubation in HTF or Ham's F-10 medium, whereas the mean linearity was decreased significantly. Follicular fluid and GC cultures significantly increased hyperactivation and the acrosome reaction compared with the values obtained using HTF and Ham's F-10 medium. Progesterone and 17OH-progesterone levels were increased significantly after incubation in FF and GC cultures compared with HTF and Ham's F-10 medium. Conclusion(s): Follicular fluid and GC cultures increase sperm motility parameters, hyperactivation, and the acrosome reaction. This effect may be related to GC detoxification of the microenvironement or GC secretion of peptides, glycoproteins, growth factors (insulin-like growth factors 1 and 2), or steroids (progesterone and 17OH-progesterone).

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. Morotti E, Persico N, Battaglia B, Fabbri R, Meriggiola MC, Venturoli S, and Battaglia C. Body imaging and sexual behavior in lean women with polycystic ovary syndrome. J Sex Med 2013;10:2752–2760.

To compare the effects of depot and daily forms of GnRH analogs in IVF programs. One hundred seventeen patients undergoing IVF, with no severe male factor, were randomized between two treatment groups. Pituitary desensitization was obtained in group 1 (60 patients) with a single IM injection of leuprorelin (3.75 mg), and in group 2 (57 patients) with buserelin (0.3 mg SC twice daily). In a subgroup of 10 patients (5 for the depot form and 5 for the daily form) several GnRH tests were performed to investigate pituitary desensitization. No differences were found in the time to reach desensitization. Resumption of pituitary activity occurred in 7 days with the daily form and in about 2 months with the depot form. No significant differences were found in the stimulation pattern, oocyte quality, percentage of fertilization. The pregnancy rate per transfer was slightly, but not significantly, better in the depot group (29.4% vs 25.9%). Implantation rate (11.9% vs 12.3%) and the percentage of miscarriages (26.6% vs 28.5%) were similar. Depot and daily forms of GnRH analogs are equally effective in superovulation induction for IVF. Considering improved patient compliance and preference, depot forms are advantageous.

Cancer treatments improve the survival rate of children and adolescents; however chemo- and radiotherapy result in gonadal damage leading to acute ovarian failure and sterility. Ovarian tissue cryopreservation allows long-term storage of primordial follicles and represents the only possibility of preserving the potential fertility in prepubertal girls. The aim of the present study is to describe our experience in ovarian tissue cryopreservation in 45 pediatric patients. The number of follicles per square millimeter of the overall section area and follicle quality were evaluated histologically. A strong negative correlation was found between age and follicular density in patients both prior to and after chemotherapy ( 𝑃 < 0 . 0 0 0 1 ). Damage in follicular quality, that is, increased oocyte vacuolization and detachment of the oocyte from granulosa cells, was found after chemotherapy. Ovarian tissue cryopreservation, preferably performed before initiation of chemotherapy, should be offered to pediatric patients, including prepubertal girls, at risk of sterility.

Objective To compare the effects of depot and standard release form of triptoreline in superovulation induction in IVF programs. Design One hundred ninety-six patients undergoing IVF were randomized between two treatment groups. Pituitary desensitization was obtained in group 1 (102 patients) with a single IM injection of 3.75mg D-Trp-6-luteinizing hormone-releasing hormone (LH-RH) and, in group 2 (94 patients), with daily SC administration of 0.1mg D-Trp-6-LH-RH. In a subgroup of 11 patients, a series of GnRH tests was performed to investigate pituitary desensitization and, in another subgroup of 12 patients, a study of luteal phase steroid profile was performed. In an additional 23 patients, a series of GnRH tests were performed to investigate pituitary desensitization during the late follicular and midluteal phases. Results No differences were found in the time necessary to reach desensitization (11.3±1.03 versus 11.3±1.45days; mean±SEM), whereas resumption of pituitary activity takes place in 7days after the discontinuation of the daily form and in about 2months after discontinuation of the depot form. No differences were found in the duration of stimulation, number of FSH ampules, E 2 levels, and number of follicles (11.7±0.68. versus 12.2±0.68) on hCG administration day and the total oocytes collected (9.1±0.6 versus 9.2±0.64). Oocyte quality, percentage of fertilization and cleavage, pregnancy rate per transfer (28.7% versus 25.6%), and miscarriages (about 30%) were similar in the two protocols. No difference was found in hormonal levels during the luteal phase. In both groups there was a high incidence of multiple pregnancy. Conclusion Comparable results can be achieved with both long-acting and standard-release forms of GnRH analogs in patients undergoing assisted reproduction in terms of follicular stimulation and abortion rates despite differences in the duration of pituitary suppression.

Skeletal maturity was studied in 82 healthy adolescent girls age between 12 and 19 who had reached the menarche from 0.5 to 5 years previously. 56 subjects (68.2%) had closed epiphyses while they were open in 26 (31.8%). The incidence of open epiphyses was 61% in the first year after the menarche and progressively decreased showing negative correlation with time elapsed since the menarche (r = 0.98, P0.0001). The mean +/- SE height increased from 158.7 +/- 1.3 cm in the first year after the menarche, to 162.7 +/- 1.3 cm in the fifth year after the menarche. No significant differences were seen in estradiol, estrone, testosterone, dihydrotestosterone and androstenedione levels between subjects with open or closed epiphyses while dehydroepiandrosterone and its sulfate levels were higher in the closed epiphyses group. Girls with open epiphyses at first evaluation grew about 3.5 cm in the next three years.

The long-term effects and tolerability of flutamide (Flu) in women with acne and seborrhea were evaluated. Over the 15-year period, which spans from January 1991 to January 2006, a total of 230 Caucasian women with acne, 211 of which also with seborrhea, received yearly reducing doses (250, 125, and 62.5 mg/day) of Flu alone or combined with oral contraceptive for a period varying from 3 to 6 years and more. Clinical and endocrine evaluations were performed half-yearly and yearly, respectively, in the first 3 years of the study, and yearly in the following years. Liver function evaluations were performed quarterly. Both the groups of patients under Flu therapy showed a similar, marked decrease in acne and seborrhea scores after 6 months of treatment compared with basal values. The maximum drug effect was observed after 1 year, and it was maintained during the following years of treatment. Androgens were strongly suppressed during treatment. During the first year of treatment with 250 mg/day, 4.8% of patients interrupted the study due to hepatic disorders. During the following years on the 125 and 62.5 mg/day doses, no transaminase elevation was observed. The study supports very low-dose Flu as a first-line therapy for women with acne and seborrhea.