Optimization of Platelet-Rich Plasma Preparation for Regenerative Medicine: Comparison of Different Anticoagulants and Resuspension Media.

Platelet-rich plasma (PRP) has emerged as a promising therapy in regenerative medicine. However, the lack of standardization in PRP preparation protocols presents a challenge in achieving reproducible and accurate results. This study aimed to optimize the PRP preparation protocol by investigating the impact of two different anticoagulants, sodium citrate (SC) and ethylenediaminetetraacetic acid (EDTA), and resuspension media, plasma versus sodium chloride (NaCl). Platelet recovery rates were calculated and compared between groups, in addition to platelet activity and vascular endothelial growth factor (VEGF) released into plasma after PRP activation. The platelet recovery rate was higher with EDTA in comparison to SC (51.04% vs. 29.85%, p = 0.005). Platelet activity was also higher, with a higher expression of two platelet antibodies, platelet surface P-Selectin (CD62p) and PAC-1, in the EDTA group. The concentration of VEGF was higher with SC in comparison to EDTA (628.73 vs. 265.44 pg/mL, p = 0.013). Platelet recovery rates and VEGF levels were higher in PRP resuspended in plasma when compared to NaCl (61.60% vs. 48.61%, p = 0.011 and 363.32 vs. 159.83 pg/mL, p = 0.005, respectively). Our study reinforces the superiority of EDTA (as anticoagulant) and plasma (for resuspension) in obtaining a higher platelet recovery and preserving platelet functionality during PRP preparation.

Recent insights explaining susceptibility to endometriosis-From genetics to environment.

Endometriosis is a disease with a heterogeneous pathogenesis, explained by multiple theories, and also with a polymorphic presentation. The purpose of this literature review is to systematize the genetic, inflammatory, and environmental factors related to the pathophysiology of endometriosis. Current evidence suggests that endometriosis is a complex inherited genetic condition, in which the genes that determine susceptibility to the disease interact with the environment to develop different phenotypes. Genetic variants associated with risk of endometriosis have been identified in several genome-wide association studies, in addition to a group of genes related to the pathophysiology of endometriosis, namely the estrogen, progesterone and androgen receptors and the cytochrome P450 gene, as well as the p53 gene. The role of inflammation is controversial; however, it is an essential process, both in the initiation and perpetuation of the disease, in and outside the pelvis. Alterations in reactive oxygen species pathways that consequently determine oxidative stress are typical in the inflammatory environment of endometriosis. The role of environmental factors is a relatively new and broad-spectrum topic, with inconsistent evidence. Multiple factors have been studied such as endocrine-disrupting chemicals, metals, intrauterine exposure to diethylstilbesterol and lifestyle risk factors. In conclusion, endometriosis remains a mysterious condition, with multifactorial factors involved in its pathophysiology. The progress that has been made in the genetic predisposition to endometriosis may allow the establishment of new therapeutic targets. On the other hand, understanding the role of the environment in this disease may allow preventive intervention, minimizing its incidence and/or severity. This article is categorized under: Reproductive System Diseases > Molecular and Cellular Physiology Reproductive System Diseases > Environmental Factors Reproductive System Diseases > Genetics/Genomics/Epigenetics.

Low responders may benefit from undergoing ovarian stimulation with a long GnRH agonist protocol with corifollitropin alfa followed by hMG.

OBJECTIVE: To evaluate the outcomes of a long GnRH agonist protocol with corifollitropin alfa followed by hMG in low responders. METHODS: Retrospective cohort study. Patients with a suboptimal previous ovarian response (<9 oocytes) and a normal ovarian reserve (Poseidon groups 1 and 2) were classified in 1) Group 1 (n=88), submitted to a second cycle with a GnRH antagonist protocol using rFSH/hMG; 2) Group 2 (n=66), submitted to a long GnRH agonist protocol with corifollitropin alfa followed by hMG (named as simplified long protocol). Clinical outcomes were compared between groups and between the first/second cycle of each group. RESULTS: Clinical outcomes were similar between groups. There were no differences in the number of oocytes [7(5-11.75) versus 7(5-10), p=0.802], clinical pregnancy (19.3% versus 18.2%, p=0.858) and live birth rates (18.2% versus 15.2%, p=0.619). However, baseline characteristics were different, decoding a poor prognosis among women in group 2. Both groups (1 and 2) had significantly higher number of oocytes, pregnancy, and live birth rates in the second cycle. In group 2, there was a higher rate of embryo transfer (56.1% versus 27.3%, p<0.001). In group 1, despite the similar rate of embryo transfer, there was a higher positive hCG (23.9% versus 8.0%, p=0.004). CONCLUSIONS: Both simplified long protocol and GnRH antagonist protocol are suitable for low responders. The best second cycle clinical outcomes experienced in a population with worse prognosis (group 2) suggests that the simplified long protocol may be a better option, although prospective well-conducted studies must explore this hypothesis.

Medical Grade Honey as a Promising Treatment to Improve Ovarian Tissue Transplantation.

Ovarian tissue cryopreservation is a female fertility preservation technique that presents major challenges for the maintenance of follicular viability after transplantation. The aim of this study was to evaluate and compare the application of L-Mesitran Soft®, a product containing 40% medical grade honey (MGH), with other strategies to improve ovarian grafts' viability. For this purpose, bovine ovarian tissue was vitrified, warmed and randomly assigned to culture groups: (1) control, (2) MGH 0.2% in vitro, (3) MGH in vivo (direct application in the xenotransplantation), (4) vascular endothelial growth factor (VEGF 50 ng/mL) and (5) vitamin D (100 Nm), during a 48 h period. A sixth group (6) of fragments was thawed on transplantation day and was not cultured. The tissue was xenotransplanted into immunodeficient (Rowett nude homozygous) ovariectomized rats. Grafts were analyzed 48 h after culture, and 7 and 28 days after transplantation. The tissue was subjected to histological and immunohistochemical analysis. Treatments using MGH showed the highest angiogenic and cell proliferation stimulation, with cellular apoptosis, within a healthy cellular turnover pathway. In conclusion, MGH should be considered as a potentially effective and less expensive strategy to improve ovarian tissue transplantation.

Fertility preservation in women with haematological malignancies.

The incidence of haematological malignancies is increasing in women of childbearing age. Survival rates accompany this increase, making it essential to assess the impact of treatments on their future quality of life, evaluate the impact of each treatment on ovarian reserve and define the fertility preservation techniques used by women with haematologic malignancies. A retrospective study was conducted after data collection from 61 women diagnosed with haematological malignancies and followed-up in a fertility preservation centre between January 2008 and June 2019. Cancer treatments caused a decrease in ovarian reserve, demonstrated by an increase in FSH levels and a decrease in AMH levels. When assessing which treatments have the greatest impact on AMH levels, we found that the BEACOPP regimen, and the agents vincristine, etoposide, procarbazine, prednisone and the haematopoietic stem cell transplantation were mainly responsible. Regarding pregnancy after oncological treatments, of the eleven women who became pregnant, ten did so spontaneously. This study reinforces the importance of referring patients to a fertility preservation consultation before starting oncological treatment, as most of them opt to preserve fertility. This work also helps to clarify the impact of each chemotherapeutic agent on the ovarian reserve.

The challenge of ovarian tissue culture: 2D versus 3D culture.

BACKGROUND: Cryopreservation of ovarian tissue is a powerful technique for preserving female fertility, as it can restore fertility and endocrine function. To increase the longevity of the transplant and decrease the risk of reimplantation of neoplastic cells, several studies have been carried out with culture of ovarian tissue. The aim of this study was to compare a conventional (2D) culture with an alginate matrix three-dimensional (3D) model for ovarian tissue culture. RESULTS: The ovarian tissue culture within the alginate matrix (3D) was similar to 2D culture, regarding follicular density and cell apoptosis in follicles and stroma. The proliferation rate remained stable in both models for follicles, but for stromal cell proliferation it decreased only in 3D culture (p = 0.001). At 24 h of culture, cytotoxicity was lower in the 3D model (p = 0.006). As culture time increased, cytotoxicity seemed similar. Degradation of the tissue was suggested by the histological score analysis of tissue morphology after 72 h of culture. Tissue injury was greater (p = 0.01) in 3D culture due to higher interstitial oedema (p = 0.017) and tissue necrosis (p = 0.035). CONCLUSION: According to our results, 3D culture of ovarian tissue has no advantage over 2Dculture; it is more time consuming and difficult to perform and has worse reproducibility.

Providing new insights into the endometriosisassociated cancer arising in episiotomy scars.

Endometriosis-associated malignancy in an episiotomy scar is rare. The predictive factors are poorly understood as are the mechanisms and pathways associated with implantation and malignant transformation. In this study we describe the cases reported in the literature of malignancies arising in endometriosis foci of an episiotomy scar. We identified 5 cases described between 1990 and 2016. These cases represent recurrence of endometriotic lesions in an episiotomy scar after previous diagnosis of endometriosis, 3 to 25 months before. Histology revealed clear cell tumours in 4 cases and a serous papillary carcinoma. The approach encompassed surgical removal for diagnosis and as part of the therapeutic strategy. Adjuvant treatment was performed depending on classical prognostic factors. Mechanisms of endometriosis implantation in scars include the influence of estrogens in the healing process and activation of COX-2, aromatase and matrix metalloproteinases. Nevertheless, for malignant transformation, other pathways seem to play a role, namely inflammation, immune response and oxidative stress, induced by iron deposits due to haemorrhage. Further studies are needed to allow the establishment of a predictive model for malignant transformation of endometriosis in episiotomy scars.

Impact of Surgical Management of Endometrioma on AMH Levels and Pregnancy Rates: A Review of Recent Literature.

Ovarian endometrioma are found in up to 40% of women with endometriosis and 50% of infertile women. The best surgical approach for endometrioma and its impact on pregnancy rates is still controversial. Therefore, we conducted a literature review on surgical management of ovarian endometrioma and its impact on pregnancy rates and ovarian reserve, assessed by anti-Müllerian hormone (AMH) serum levels. Ovarian cystectomy is the preferred technique, as it is associated with lower recurrence and higher spontaneous pregnancy rate. However, ablative approaches and combined techniques are becoming more popular as ovarian reserve is less affected and there are slightly higher pregnancy rates. Preoperative AMH level might be useful to predict the occurrence of pregnancy. In conclusion, AMH should be included in the preoperative evaluation of reproductive aged women with endometriosis. The surgical options for ovarian endometrioma should be individualized. The endometrioma ablation procedure seems to be the most promising treatment.

Fertility preservation in ovarian cancer patients.

Ovarian cancer is more frequent in post-menopausal women, however it can also occur in young premenopausal women. After diagnosis and cancer staging, health care providers should address the possibility of infertility and might offer fertility preservation options. Chemotherapy, frequently used when treating ovarian cancer, has proven to cause extensive ovarian damage. Standard surgery may be aggressive and the recurrence risk may not be relevant enough to opt for these approaches. Fertility sparing surgery has been progressively accepted and many alternative surgical approaches have proven to be successful in both cancer treatment, fertility preservation and low recurrence rates. Though there are several techniques available for fertility preservation in cancer patients, when dealing with ovarian cancer patients, oocyte and embryo cryopreservation are the only suitable. Ovarian tissue cryopreservation has been largely studied, but no data on ovarian cancer patients exist, due to the risk of reimplanting cancer cells.

Prevalence of cytogenetic abnormalities and FMR1 gene premutation in a Portuguese population with premature ovarian insufficiency.

INTRODUCTION: Chromosome abnormalities contribute to about 10% of cases of premature ovarian insufficiency. Most are associated with X chromosome. Fragile mental retardation 1 (FMR1) gene premutation has an estimated prevalence of 1% - 7% in sporadic cases and up to 13% in familial cases. Our aim was to describe the clinical characteristics, cytogenetic and FMR1 testing of a Portuguese population with premature ovarian insufficiency. MATERIAL AND METHODS: Women diagnosed with premature ovarian insufficiency in a Portuguese tertiary centre were retrospectivelyanalysed. Data were retrieved from electronic medical records including clinical characteristics, cytogenetic and FMR1 testing. The main outcome measures were the prevalence of chromosome abnormalities and FMR1 premutation in a Portuguese population with premature ovarian insufficiency. RESULTS: Ninety-four patients were included, with a median age at menopause of 36 years. The prevalence of chromosome abnormalities was 16.5% (14/85) and most were X chromosome related (78.6%). The prevalence of FMR1 premutation was 6.7% (6/90). The prevalence of karyotypic abnormalities or FMR1 premutation did not differ significantly between familial and sporadic cases. Neither chromosome abnormalities nor FMR1 premutation influenced age at menopause or follicle stimulating hormone levels at diagnosis in premature ovarian insufficiency patients. DISCUSSION: This is the first study describing the clinical characteristics and both cytogenetic and FMR1 testing in a Portuguese population with premature ovarian insufficiency. The rate of chromosome abnormalities in our sample was higher than in other populations, while the prevalence of FMR1 premutation was similar to previous reports. CONCLUSION: Our results underline the importance of genetic screening in premature ovarian insufficiency patients in both etiological study and genetic counselling.

Introdução: As anomalias cromossómicas contribuem para 10% dos casos de insuficiência ovárica prematura estando maioritariamente associadas ao cromossoma X. A pré-mutação do gene fragile mental retardation 1 (FMR1) tem uma prevalência estimada de 1% - 7% nos casos esporádicos e até 13% nos casos familiares. O nosso objetivo foi descrever as características clínicas e a análise citogenética e do gene FMR1 de uma população Portuguesa com insuficiência ovárica prematura. Material e Métodos: Análise retrospetiva das mulheres com o diagnóstico de insuficiência ovárica prematura vigiadas num hospital terciário Português. Recolha de dados através do processo médico eletrónico incluindo características clínicas, análise citogenética e análise do gene FMR1. Os desfechos principais foram a prevalência de anomalias cromossómicas e da pré-mutação FMR1 numa população Portuguesa com insuficiência ovárica prematura. Resultados: Foram incluídas 94 doentes, com uma mediana de idade de menopausa de 36 anos. A prevalência de anomalias cromossómicas foi 16,5% (14/85) e a maioria estavam relacionadas com o cromossoma X (78,6%, n = 11). A prevalência da pré-mutação FMR1 foi de 6,7% (6/90). A prevalência de anomalias cromossómicas ou pré-mutação FMR1 não diferiu entre casos esporádicos e familiares. Nem as anomalias cromossómicas nem a pré-mutação FMR1 influenciaram a idade de menopausa ou os níveis da hormona estimulante dos folículos capilares aquando do diagnóstico na população com insuficiência ovárica prematura. Discussão: Este é o primeiro estudo a descrever as características clínicas e a análise citogenética e do gene FMR1 numa população Portuguesa com insuficiência ovárica prematura. A prevalência de anomalias cromossómicas na nossa amostra foi superior à descrita para outras populações, enquanto a prevalência da pré-mutação FMR1 foi semelhante à descrita em estudos anteriores. Conclusão: Os nossos resultados sublinham a importância do rastreio genético em doentes com insuficiência ovárica prematura, quer no estudo etiológico, quer no aconselhamento genético.

Fertility preservation with successful pregnancy outcome in a patient with transplanted heart and non-Hodgkin's lymphoma - a case report.

BACKGROUND: Fertility preservation must be discussed with reproductive age women before cancer treatment. Heart transplantation raises complex issues in pregnancy. Pregnancy in a heart transplant woman after pelvic irradiation involves close multidisciplinary follow-up to avoid complications in the mother and the foetus. We report the first live birth in a heart transplant woman after pelvic irradiation, chemotherapy and fertility preservation. CASE PRESENTATION: A 36-year-old heart transplant woman with pelvic non-Hodgkin lymphoma spared her fertility, with cryopreservation of oocytes and embryos, before chemotherapy and pelvic irradiation. After multidisciplinary discussion and pre-conception evaluation, pregnancy was achieved. A close follow-up by a multidisciplinary team allowed a normal pregnancy without maternal or foetal complications and the delivery of a healthy infant. CONCLUSIONS: Achieving pregnancy in heart transplant women with iatrogenic ovarian failure after oncologic treatment including pelvic irradiation is possible and can be successful. Careful and close surveillance by a multidisciplinary team is mandatory due to increased risk of maternal and foetal complications.

Mirror syndrome assocciated with patau syndrome: A case report / Síndrome de espelho associada a uma síndrome de Patau: um relato de caso

Abstract Mirror syndrome is an unusual pathological condition in which maternal edema in pregnancy is seen in association with severe fetal and/or placental hydrops. The disease can be life-threatening for both the mother and the fetus. The pathogenesis is poorly understood, and may be confused with preeclampsia, even though distinguishing features can be identified. We report a rare case of mirror syndrome with maternal pulmonary edema associated with fetal hydrops due to Patau syndrome.

Resumo A síndrome de espelho é uma patologia invulgar na qual o edemamaterno é observado em associação com hidropsia fetal e/ou placentária graves. Esta doença pode ser fatal paraamãe e para o feto. A sua patogênese émal compreendida, e pode ser confundida compré-eclâmpsia,mesmo comcaracterísticas distintivas identificadas. Relatamos um caso raro de síndrome de espelho com edema pulmonar materno associado a hidropsia fetal devido a síndrome de Patau.

Mirror Syndrome assocciated with Patau Syndrome: A Case Report.

Mirror syndrome is an unusual pathological condition in which maternal edema in pregnancy is seen in association with severe fetal and/or placental hydrops. The disease can be life-threatening for both the mother and the fetus. The pathogenesis is poorly understood, and may be confused with preeclampsia, even though distinguishing features can be identified. We report a rare case of mirror syndrome with maternal pulmonary edema associated with fetal hydrops due to Patau syndrome.

A síndrome de espelho é uma patologia invulgar na qual o edema materno é observado em associação com hidropsia fetal e/ou placentária graves. Esta doença pode ser fatal para a mãe e para o feto. A sua patogênese é mal compreendida, e pode ser confundida com pré-eclâmpsia, mesmo com características distintivas identificadas. Relatamos um caso raro de síndrome de espelho com edema pulmonar materno associado a hidropsia fetal devido a síndrome de Patau.