Dosimetric evaluation of ovaries and pelvic bones associated with clinical outcomes in patients receiving total body irradiation with ovarian shielding.

Total body irradiation (TBI) with ovarian shielding is expected to preserve fertility among hematopoietic stem cell transplant (HSCT) patients with myeloablative TBI-based regimens. However, the radiation dose to the ovaries that preserves ovarian function in TBI remains poorly understood. Furthermore, it is uncertain whether the dose to the shielded organs is associated with relapse risk. Here, we retrospectively evaluated the relationship between fertility and the dose to the ovaries, and between relapse risk and the dose to the pelvic bones. A total of 20 patients (median age, 23 years) with standard-risk hematologic diseases were included. Median follow-up duration was 31.9 months. The TBI prescribed dose was 12 Gy in six fractions for three days. Patients' ovaries were shielded with cylinder-type lead blocks. The dose-volume parameters (D98% and Dmean) in the ovaries and the pelvic bones were extracted from the dose-volume histogram (DVH). The mean ovary Dmean for all patients was 2.4 Gy, and 18 patients recovered menstruation (90%). The mean ovary Dmean for patients with menstrual recovery and without recovery were 2.4 Gy and 2.4 Gy, respectively, with no significant difference (P = 0.998). Hematological relapse was observed in five patients. The mean pelvis Dmean and pelvis D98% for relapse and non-relapse patients were 11.6 Gy and 11.7 Gy and 5.6 Gy and 5.3 Gy, respectively. Both parameters showed no significant difference (P = 0.827, 0.807). In conclusion, TBI with ovarian shielding reduced the radiation dose to the ovaries to 2.4 Gy, and preserved fertility without increasing the risk of relapse.

Combination of medical and surgical management in successful treatment of caesarean scar pregnancy: a case report series.

BACKGROUND: There is no clear consensus on the management of caesarean scar pregnancy (CSP), a complex and life-threatening condition. The objective of this study was to present a novel approach to management of CSP that combines medical therapy of multidose methotrexate and mifepristone with active surgical management by uterine curettage and consecutive local haemostasis. CASE PRESENTATION: We report on a prospective case series of six women with first trimester pregnancy, in whom the diagnosis of CSP was confirmed by 2D and color Doppler transvaginal ultrasound and serial hormone chorionic gonadotropin (hCG) testing. Women were between 23 and 36 years old and had at least one previous delivery by caesarean. At admission, gestational age ranged between 6 to 14 weeks, and serum hCG levels between 397 and 23,000 mUI/ml. Upon decision of pregnancy termination, medical management was undertaken in all cases and 1 mg/kg systemic Methotrexate was administered between 1 and 5 daily doses. Mifepristone was part of the treatment in cases with live pregnancy. Surgical management was employed for the cases were an embryo was seen by ultrasound, being prompted by inadequate response to Methotrexate and/or signs of miscarriage with vaginal bleeding. Curettage combined with local isthmic balloon or vaginal pack tamponade prevented further complications. High treatment rates with preservation of fertility was achieved in all patients except one who underwent hysterectomy for invasive placentation. Ultrasound and hCG levels surveillance ensured that the resolution of pregnancy was achieved. CONCLUSION: Women with history of delivery by caesarean section should be carefully monitored in future pregnancies for prompt diagnosis of CSP. Early diagnosis of CSP allows selection of successful conservative therapy. Through this case series we contribute with our experience to the body of knowledge about the management of this serious complication of early pregnancy.

Ultrasound-guided follicle aspiration at time of laparotomy in a patient with Mayer-Rokitansky-Küster-Hauser syndrome.

OBJECTIVE: To demonstrate a unique case of direct ultrasound-guided follicle aspiration at time of a laparotomy in a patient with Mayer-Rokitansky-Küster-Hauser Syndrome. DESIGN: Video presentation. SETTING: Academic fertility center PATIENT(S): A 32-year-old gravida 0 with Mayer-Rokitansky-Küster-Hauser Syndrome and bilateral ovarian masses presented as a fertility preservation consult from gynecologic oncology due to the possibility of bilateral oophorectomy. Due to the appearance and size of the left ovary oncology planned to perform an exploratory laparotomy and left oophorectomy, with possible right oophorectomy. The patient and her partner desired embryo cryopreservation with plans for future use in a gestational carrier. She had previously undergone vaginal dilator therapy, however her ovaries were inaccessible transvaginally due to their cephalad location and small caliber of the vaginal pouch. The plan was made to proceed with controlled ovarian stimulation and concurrent ultrasound-guided follicle aspiration of the right ovary at the time of laparotomy following left oophorectomy. INTERVENTION(S): Ultrasound-guided follicle aspiration in vivo at time of laparotomy. MAIN OUTCOME MEASURE(S): Successful controlled ovarian stimulation, oocyte retrieval and embryo cryopreservation. RESULT(S): The patient underwent a long agonist protocol and received a total of 2,525 units of gonadotropin with a peak estradiol of 3,264 pg/ml. She required a total of 9 days of stimulation. The normal right ovary responded as expected, and the left ovary remained unchanged. Following laparotomy and left oophorectomy, direct application of the transvaginal ultrasound probe was used to aspirate all visible follicles on the right side in vivo. Twenty-four oocytes were retrieved, 15 were mature and 5 blastocysts were cryopreserved. Final pathology of left ovary returned as serous cystadenoma. The right ovary was examined by gynecologic oncology prior to and following retrieval and was thought to be normal and remained in situ. CONCLUSION(S): Although the approach described here is not feasible in most cases, this video demonstrates a unique and successful fertility preservation technique by direct ultrasound-guided follicle aspiration in vivo at the time of laparotomy in a Mayer-Rokitansky-Küster-Hauser Syndrome patient and to our knowledge, is the first description of its kind. This retrieval would have otherwise been limited by lack of access transvaginally and limited visualization transabdominally. This combined approach should be considered in future patients with müllerian anomalies and similar complicating factors necessitating laparotomy.

Using microscope for onco-testicular sperm extraction for bilateral testis tumors.

OBJECTIVE: To demonstrate a step-by-step approach to the use of the operating microscope for onco-testicular sperm extraction. DESIGN: Video presentation. SETTING: University hospital. PATIENT(S): A 34-year-old man (status post right orchiectomy at another institution for pT3 pure seminoma with negative preoperative tumor markers) was referred for contralateral orchiectomy for multifocal left testis mass and fertility preservation. A postoperative semen analysis for attempted cryopreservation of ejaculated semen identified azoospermia. INTERVENTION(S): Left radical orchiectomy, left microsurgical onco-testicular sperm extraction (TESE). MAIN OUTCOME MEASURE(S): Intraoperative technique with commentary highlighting tips for successful fertility preservation via microsurgical onco-TESE. Discussion of alternatives. RESULT(S): This video provides a step-by-step guide to microsurgical onco-TESE coordinated with radical orchiectomy for testis cancer as a means of fertility preservation in an azoospermic patient. Preoperative imaging with scrotal ultrasound can serve as a useful guide for targeting microdissection to areas of normal testicular parenchyma for extraction of seminiferous tubules likely to host normal spermatogenesis. This patient had successful recovery and cryopreservation of abundant testicular sperm following targeted ex-vivo testicular microdissection. CONCLUSION(S): Microsurgical onco-TESE may be offered to azoospermic patients when undergoing orchiectomy for testis cancer. Use of preoperative imaging and the surgical microscope guide surgical dissection and optimize sperm recovery.

Vitrification of testicular tissue from prepubertal cats in cryotubes using different cryoprotectant associations.

Protocols for the cryopreservation of testicular tissue are not yet established. In cats, few studies have been conducted on testicular vitrification using different cryoprotectant associations (CPAs). Thus, the objective of this study was to compare the effect of different CPAs on the vitrification of testicular tissue from prepubertal cats in cryotubes. We used 10 pairs of testicles, with each pair divided into 8 fragments that were distributed into different experimental groups. Two of these fragments were allocated into the control group (CG) and the other six were distributed according to the CPAs to be tested (dimethyl sulfoxide (DMSO)/glycerol (GLY), ethylene glycol (EG)/GLY, or DMSO/EG). The cryoprotectants were used at a final concentration of 5.6 M. The fragments were subjected to vitrification in cryotubes and after 1 week, they were warmed and processed for histomorphologic assessment, quantification of nucleolar organizer regions (NORs), and determination of cell viability. The DMSO/EG and EG/GLY groups presented the greatest cell separation from the cell basement membrane and the highest degrees of retraction of the basal membrane. In these aspects, DMSO/GLY did not differ from the CG and both were significantly superior to the other groups. In terms of cell distinction, visibility of the nucleus, and nuclear condensation, all the vitrified groups had significantly lower values than the CG, while the DMSO/GLY and EG/GLY groups did not differ between themselves. Through the quantification of NORs, the potential for cell proliferation of the CG was found to have a mean of 3.80, while DMSO/GLY presented a mean of 3.60, and thus there was no significant difference between these two groups. The proliferation potentials of both groups were significantly superior to that of the DMSO/EG (mean: 2.07) and EG/GLY (mean: 1.98) groups. In the CG and DMSO/GLY group, 91.8% and 64.2% of cells, respectively, were found to be viable. The cell viabilities of both groups were significantly superior to those of DMSO/EG (52.5%) and EG/GLY (57.10%). Vitrification in cryotubes combined with the use of the DMSO/GLY association was effective in maintaining the histomorphology, cell proliferation potential, and cell viability of testicular tissue from prepubertal cats after cryopreservation.

Preservação da fertilidade masculina no Centro Hospitalar de Vila Nova de Gaia/Espinho EPE - 17 anos de experiência / Preserving male fertility at the Centro Hospitalar de Vila Nova de Gaia/Espinho EPE -17 years of experience

Objetivo. Reportar a nossa experiência nos últimos 17 anos na preservação da fertilidade masculina por doença oncológica, genética, imunológica e endocrinológica, assim como por alterações graves da espermatogénese. Material e métodos. avaliação de parâmetros demográficos, características do esperma e resultados obtidos com amostras descongeladas em ciclos de procriação medicamente assistida (PMA). Resultados. Duzentos e setenta e um homens com uma idade média de 31,5±6,3 anos (16‐52 anos) foram referenciados para PF. A doença oncológica foi a causa mais comum (34,3%), seguida de azoospermia secretora ou não obstrutiva, oligoastenoteratozoospermia (TAO) grave, doença genética, hipogonadismo/atrofia testicular, doença auto‐imune, doença endócrino‐metabólica e pré‐cirurgia potencialmente comprometedora da fertilidade. Dentro da doença oncológica, o tumor do testículo foi a neoplasia mais comum (64,5%), seguido do cancro hematológico e de etiologias variadas. Na doença genética, a síndrome de Klinefelter foi a condição mais frequente (50%). No total 194 homens ficaram com amostras criopreservadas (1.099 amostras no total; 5,7±3,4 por paciente). Os homens com doença oncológica tiveram, em média, um número significativo superior de amostras congeladas (p<0,001). Os casos de tumor do testículo apresentaram uma concentração média de espermatozoides/ml significativamente inferior face aos outros tumores (p<0,005). Durante estes 17 anos, 58 homens (29,9%) procuraram o centro para realização de técnicas de PMA com amostras descongeladas. Foram realizados 87 ciclos, com 19 gestações (21,8%). Nasceram 15 recém‐nascidos (RN) saudáveis. Conclusões. Ainda que o número de homens que tenham procurado o uso das amostras armazenadas, não se pode desvalorizar o impacto psicológico positivo da PF, tendo em conta que pode ser a única possibilidade de paternidade biológica (AU)

Objective. A report is presented of 17 years of experience in male fertility preservation due to oncological, genetic, immunological and endocrinological disease, as well as severe spermatogenesis alterations. Material and methods. An evaluation is made of demographic parameters, sperm characteristics and results of assisted reproduction techniques cycles with thawed samples. Results. A total of 271 men with a mean age 31.5±6.3 years (16 to 52 years) were referred to sperm banking. The most common cause was oncological disease (34.3%), followed by secretory azoospermia, oligoasthenoteratozoospermia, genetic disease, testicular atrophy/hypogonadism, auto‐immune disease, endocrine‐metabolic disease, and prior to potentially fertility damage surgery. Among oncological disease, testicular tumour was the most common cancer (64.5%), followed by haematological cancer, and other aetiologies. Klinefelter syndrome was the most common (50%) among the genetic diseases. A total of 194 men had samples cryopreserved (1099 total samples; 5.7±3.4 per patient). Men with oncological disease had a significantly higher number of cryopreserved samples (p<.001). Testicular tumour cases had a mean lower sperm concentration relative to other tumours (p<.005). During these 17 years, 58 men (29.9%) asked to have assisted reproduction techniques performed with their thawed samples. The 87 cycles performed resulted in 19 pregnancies (21.8%) and 15 healthy newborns. Conclusions. Although the number of men asking to use their cryopreserved samples is low, we cannot undervalue the positive psychological impact of fertility preservation, taking in consideration that it can be the only possibility to biological fatherhood (AU)

Short-term preservation of Pecari tajacu ovarian preantral follicles using phosphate buffered saline (PBS) or powdered coconut water (ACP(r)) media / Preservação de folículos ovarianos pré-antrais de Pecari tajacu por curtos períodos utilizando meios à base de solução salina fosfatada tamponada (PBS) ou água de coco em pó (ACP(r))

We compare protocols for the short-term preservation of collared peccarie's ovarian preantral follicles (PFs) by using phosphate buffered saline- (PBS) or powdered coconut water- (ACP(r)) based medium. For morphology analysis each pair of ovaries collected from six females was divided into nine fragments. One fragment was destined for morphology analysis (histology and transmission electron microscopy - TEM), constituting the control group and the other fragments were placed in tubes with PBS or ACP(r), packed in 5 L Styrofoam boxes, stored for 4h, 12h, 24h, and 36h, and then analyzed. For viability analysis a pair of ovaries from two additional females was divided into nine fragments; one fragment was immediately destined for viability analysis (Trypan blue test) and the other fragments were stored as previously described, until 24h and then analyzed. After 4h storage in ACP(r) medium, the follicular integrity was similar to control (87.8% vs 94.4%, respectively); however, ultrastructural analyses revealed swollen mitochondria as the first signals of PF degeneration. It was observed that ACP(r) (66.7%) was more efficient than PBS (49.4%) to preserve the morphological integrity after 36h storage (P<0.05); however, no differences were observed on follicular viability (P>0.05). In conclusion, the use of the ACP(r) is recommended for the short-term preservation of Pecari tajacu preantral follicles...

Compararam-se protocolos para a preservação por curtos períodos de folículos ovarianos pré-antrais (PFs) de catetos, utilizando meios à base de solução salina tamponada (PBS) ou água de coco em pó (ACP(r)). Para a análise morfológica, cada par de ovários coletados de seis fêmeas foi dividido em nove fragmentos. Um fragmento foi destinado para a análise da morfologia (histologia e microscopia eletrônica de transmissão - MET), constituindo o grupo controle, e os demais fragmentos foram colocados em tubos contendo PBS ou ACP(r), acondicionados em caixas térmicas de poliestireno expandido de 5L, armazenados durante quatro, 12, 24 e 36 horas, e, então, analisados. Para a análise da viabilidade, pares de ovários de duas fêmeas adicionais foram divididos em nove fragmentos; um deles foi imediatamente destinado à análise da viabilidade (teste com azul de Trypan), os outros fragmentos foram armazenados como descrito previamente até 24h e, então, foram analisados. Após quatro horas de armazenamento em meio ACP(r), a integridade folicular foi similar ao grupo controle (87,8% vs. 94,4%, respectivamente); contudo, a análise ultraestrutural revelou mitocôndrias edemaciadas como os primeiros sinais de degeneração dos PFs. Foi observado que o ACP(r) (66,7%) foi mais eficiente do que o PBS (49.4%) em preservar a integridade morfológica após 36h (p<0,05); entretanto, nenhuma diferença foi observada para a viabilidade folicular (P>0,05). Em conclusão, o uso da ACP(r) é recomendado para a preservação por curtos períodos de folículos pré-antrais de Pecari tajacu...

First transplantation of isolated murine follicles in alginate.

AIM: Our aim is to develop an artificial ovary allowing survival and growth of isolated follicles and ovarian cells, to restore fertility in women diagnosed with pathologies at high risk of ovarian involvement. MATERIALS & METHODS: For this, alginate beads containing isolated preantral follicles and ovarian cells were autografted to immunocompetent mice. One week after grafting, the beads were invaded by proliferating murine cells (12.1%) and capillaries. RESULTS: The recovery rate of follicles per graft ranged from 0% to 35.5%. Of the analyzed follicles, 77% were Ki67-positive and 81%, TUNEL-negative. Three antral follicles were also identified, evidencing their ability to grow in the matrix. CONCLUSION: Our results suggest that an artificial ovary is now conceivable, opening new perspectives to restore fertility in women.

Linfoma primario de cérvix: gestación tras tratamiento conservador / Primary lymphoma of the cervix: pregnancy after conservative treatment

El cérvix representa una rara localización de linfomas, con una incidencia del 0,41-0,6% de linfomas extranodales. El síntoma de inicio suele ser el sangrado vaginal, siendo diagnosticado en biopsia. No existe tratamiento estándar debido a su baja incidencia. La cirugía supone la pérdida irreversible de capacidad reproductiva. Este caso apoya la posibilidad de un manejo conservador de linfoma primario de cérvix con quimioterapia y RT si la enfermedad está localizada y se desea preservar el potencial genésico de la paciente. Sin embargo, las técnicas de criopreservación de ovocitos, embriones o transposición de tejido ovárico deberían ser planteadas previo al tratamiento, debido al mal pronóstico de este sobre la fertilidad en pacientes jóvenes (AU)

The cervix is a rare localization of lymphomas, with an incidence of 0.41- 0.6% of extranodal lymphomas. The initial symptom is usually vaginal bleeding. Diagnosis is made by biopsy. There is no standard treatment due to the low incidence of this tumor. Surgery causes irreversible loss of reproductive capacity. We report a case that illustrates the possibility of conservative treatment of primary cervical lymphoma with chemotherapy and radiotherapy in local disease if the patient wishes to preserve her reproductive ability. However, the techniques of ovocyte preservation or embryo or ovarian tissue transposition should be considered before treatment, due to its negative effects on fertility in young women (AU)

The use of a metal container for vitrification of mouse ovaries, as a clinical grade model for human ovarian tissue cryopreservation, after different times and temperatures of transport.

PURPOSE: Cryopreservation of ovarian tissue is paramount for fertility preservation, with important clinical applications, especially for women suffering from an oncological condition. Several cryopreservation methodologies have been tried in search of better outcomes, especially in terms of primor-dial and primary follicles integrity post-cryopreservation. Vitrification has successfully been applied to ovarian tissue using different carriers for tissue exposure to the liquid nitrogen (LN2). METHODS: We developed an enclosed metal vessel, which has the advantage of a faster heat transfer, when in contact with LN2 avoiding at the same time, the direct contact with tissue. Additionally, we assessed the effect of different times and temperatures of transport between the collection of mouse ovaries and the beginning of cryopreservation, on follicular morphology after vitrification. RESULTS: Our results suggest that 37 °C and R.T. help to maintain normal primordial and primary follicle morphology for up to 4 hrs after collection and beginning of vitrification in a metal container. CONCLUSION: These data show that the metal container is an appropriate carrier for mouse ovary vitrification. The rate of morphologically normal primordial follicles up to 4 hrs.