Reference standards for follicular density in ovarian cortex from birth to sexual maturity.

RESEARCH QUESTION: Are age-normalized reference values for human ovarian cortical follicular density adequate for tissue quality control in fertility preservation? DESIGN: Published quantitative data on the number of follicles in samples without known ovarian pathology were converted into cortical densities to create reference values. Next, a sample cohort of 126 girls (age 1-24 years, mean ± SD 11 ± 6) with cancer, severe haematological disease or Turner syndrome were used to calculate Z-scores for cortical follicular density based on the reference values. RESULTS: No difference was observed between Z-scores in samples from untreated patients (0.3 ± 3.5, n = 30) and patients treated with (0.5 ± 2.9, n = 48) and without (0.1 ± 1.3, n = 6) alkylating chemotherapy. Z-scores were not correlated with increasing cumulative exposure to cytostatics. Nevertheless, Z-scores in young treated patients (0-2 years -2.1 ± 3.1, n = 10, P = 0.04) were significantly lower than Z-scores in older treated patients (11-19 years, 2 ± 1.9, n = 15). Samples from patients with Turner syndrome differed significantly from samples from untreated patients (-5.2 ± 5.1, n = 24, P = 0.003), and a Z-score of -1.7 was identified as a cut-off showing good diagnostic value for identification of patients with Turner syndrome with reduced ovarian reserve. When this cut-off was applied to other patients, analysis showed that those with indications for reduced ovarian reserve (n = 15) were significantly younger (5.9 ± 4.2 versus 10.7 ± 5.9 years, P = 0.004) and, when untreated, more often had non-malignant haematologic diseases compared with those with normal ovarian reserve (n = 24, 100% versus 19%, P = 0.009). CONCLUSION: Z-scores allow the estimation of genetic- and treatment-related effects on follicular density in cortical tissue from young patients stored for fertility preservation. Understanding the quality of cryopreserved tissue facilitates its use during patient counselling. More research is needed regarding the cytostatic effects found in this study.

Impact of cancer chemotherapy before ovarian cortex cryopreservation on ovarian tissue transplantation.

STUDY QUESTION: How efficacious is transplantation of ovarian cortex previously exposed to chemotherapy? SUMMARY ANSWER: Prior exposure to chemotherapy did not disrupt the function of cryopreserved ovarian tissue after transplantation. WHAT IS KNOWN ALREADY: Ovarian tissue cryopreservation (OTC) followed by ovarian tissue transplantation (OTT) is an efficacious technique for restoration of female fertility. At least 130 children have been born following this procedure. To date, little is known about the efficacy of OTT in patients exposed to cancer chemotherapy prior to OTC. STUDY DESIGN, SIZE, DURATION: This study evaluates the recovery of ovarian function and fertility in 31 consecutive patients who had received OTT, between 2005 and 2015. PARTICIPANTS/MATERIALS, SETTING, METHODS: Thirty one patients, wanting children, were transplanted with autologous ovarian cortex, among which 22 patients (71%) had been exposed to chemotherapy before OTC. Recovery of ovarian function was considered total once menstruation occurred. Ovarian function recovery (OFR), ovarian graft survival, and incidence of pregnancy were related to previous chemotherapy exposure, type of chemotherapy and graft characteristics (number of grafted fragments and follicular density). MAIN RESULTS AND ROLE OF CHANCE: The amount of ovarian tissue collected was the only parameter to show any significant change between patients with versus without previous chemotherapy. At 1 year after OTT, the cumulative incidence of OFR was 83% (93% in patients exposed to chemotherapy and 67% in others (P = 0.14)). A low follicular density (<0.3 foll/mm2) in the transplant and a low number of grafted fragments (<16) were significantly associated with a delayed OFR. Graft survival at 2 years after OTT was 77%. It was significantly lower in patients exposed to bifunctional alkylating agents before ovarian cryopreservation and in patients with a low follicular density. The proportion of women who succeeded in having at least one live birth was 23% in the total population, 0% (0/9) in the group 'no previous chemotherapy', and 32% (7/22) in the group 'previous chemotherapy'. The cumulative incidence of pregnancy (Kaplan-Meier) at 3 years after OTT was 36% overall and 49% in case of previous chemotherapy, with no difference related to previous chemotherapy exposure. In total there were 13 pregnancies and 8 births in 7 patients. LIMITATIONS, REASONS FOR CAUTION: The pathology in the two groups of patients was not comparable. In the group of patients who had chemotherapy before OTC, there were 95% of hematological malignancies. In the group of patients who did not have chemotherapy before OTC only 1 out of 9 patients had a malignant hematological disease while 44% had some pathology affecting the ovaries. Few women are available for study and only large changes are likely to have statistical significance. WIDER IMPLICATIONS OF THE FINDINGS: These results suggest that prior cancer chemotherapy should no longer be considered a limitation to cryopreservation of ovarian tissue and current recommendations in this regard should be revised. STUDY FUNDING/COMPETING INTEREST(S): This study was supported by the Agence de la Biomédecine (France's biomedical office). There are no competing interests to report. TRIAL REGISTRATION NUMBER: NCT02184806.

Assessment of reflectance confocal microscopy for non-invasive selection of optimal ovarian cortex fragments for autotransplantation.

RESEARCH QUESTION: Can reflectance confocal microscopy (RCM) be used to determine follicle density in human ovarian cortex fragments that are intended for fertility restoration? DESIGN: RCM was used on living cortex tissue fragments derived from five bovine ovaries and 13 human ovaries. All tissue fragments were cryopreserved and thawed before RCM analysis. Follicle numbers and distribution were determined by RCM and histology. Before and after RCM, general tissue viability and follicle integrity were assessed by a glucose uptake assay and neutral red staining, respectively. RESULTS: RCM can detect all stages of follicle development in living ovarian tissue to a maximum depth of 250 µm. In bovine tissue, all follicles were located within this 0-250 µm range. In human ovarian tissue, follicles were also present below the 250 µm RCM threshold, implying that only a percentage of the total number of follicles could be detected with RCM. The percentage of follicles detected by RCM appeared to be age dependent. The RCM procedure did not affect the glucose uptake by the tissue, whereas neutral red staining indicated a high level of follicle survival. CONCLUSION: In this proof of concept study, we have shown that RCM is a promising technique to determine the density of follicles ex vivo in living human ovarian cortex fragments, apparently without compromising the vitality of the tissue. Safety studies and further optimization of the RCM technique with a focus on increasing the penetration depth are required before clinical use of RCM.

Female Reproductive Performance in the Mouse: Effect of Oral Melatonin.

Although melatonin has some of the broadest ranges of actions on the physiology of vertebrates, especially on their reproductive processes, the mechanism by which melatonin regulates animal reproduction is still incompletely understood. This study was designed to determine the effect of oral melatonin on the reproductive performance of female mice. Female ICR mice (7 weeks old) were given melatonin-containing water (3, 30 and 300 µg/mL; melatonin) or water only (control) until 10 weeks of age. Then, some of the mice were successfully mated (confirmed by vaginal plugs), and the number of live births and their weights were recorded. Some mice were used for a histological analysis of the number of follicles in the ovaries. Others were used for oocyte collection after superovulation, and in vitro fertilization (IVF) was performed. The mRNA expression of the apopotosis-related genes (BAX, BCL2) in the IVF embryos were analyzed. After melatonin administration, the mice showed similar serum melatonin levels to that of the control. The number of antral follicles per mm² unit area in the 30 µg/mL melatonin-treated group (14.60) was significantly higher than that of the control (7.78), which was lower than that of the 3 µg/mL melatonin-treated group (12.29). The litter size was significantly higher in the 3 µg/mL melatonin-treated group (15.5) than in the control (14.3). After IVF, the hatched blastocyst formation rate in the 30 µg/mL melatonin-treated group (85.70%) was significantly higher than that of the control (72.10%), and it was the same for the BCL2/BAX expression ratio. Although oral melatonin did not appear to have an effect on the serum melatonin rhythm in the mouse, melatonin did increase litter size at the 3 µg/mL dose level, and improved the developmental competency of IVF embryos at the 30 µg/mL level.

Morfología de la Piel y Producción de Lana en Cruzamiento Absorbente con Merino Multipropósito / Skin Morphology and Wool Production on Merino Multipurpose from Absorbent Crosses

Las tendencias del mercado de fibras textiles muestran que las lanas finas son las que se adaptan a las preferencias de la industria textil. En Argentina, la producción de lanas finas es insignificante y esto representa una limitante para el crecimiento lanero del país. La necesidad de desarrollar una alternativa de producción ovina lleva a considerar al Merino Multipropósito como una opción interesante. En la provincia de Corrientes, se iniciaron cruzamientos con estos animales, pero existen inquietudes con respecto a lograr una disminución en el diámetro de la fibra y que repercuta en el peso del vellón. El objetivo del trabajo fue evaluar el efecto del cruzamiento de ovejas Corriedale con machos MPM, sobre las características de la piel y la producción y calidad de lana. Los parámetros evaluados fueron diámetro de fibra, densidad de folículos y relación S/P. Para el recuento de folículos primarios y secundarios se realizaron biopsias de piel con auxilio de un sacabocado. Las características en relación con la producción y calidad de lana se consideraron entre borregas contemporáneas. La comparación realizada en este trabajo permitiría atribuir la disminución del diámetro de fibra, al cruzamiento con MPM. La densidad de folículos en las madres Corriedale, en las borregas Corriedale y en la F1 (Corriedale x MPM) fueron diferente significativamente. La relación S/P fue 9,08 ± 2,32, 9,46 ± 1,57, y 11,93± 2,18, respectivamente. El peso del vellón en borregas de la F1 (Corriedale x MPM) fue inferior y significativamente diferente de sus contemporáneas Corriedale. El rinde al lavado en las borregas Corriedale fue superior al de las borregas nacidas del cruzamiento con MPM. Las diferencias fueron significativas en el largo de mecha entre borregas Corriedale y Corriedale x MPM. En las condiciones realizadas no se pudo comprobar todas las expectativas esperadas con el cruzamiento. Nuevas experiencias serán necesarias para respaldar morfológica y productivamente la posible inversión.

Textile market trends show that fine wools are more suitable to reach the preferences of textile industry. Production of fine wool in Argentina are insignificant, which is a limitation on the development of the country. The need to develop new alternatives of production leads us consider the Multipurpose Merinos as an option. Crossbreeding with these animals began at Corrientes state in order to achieve a reduction in the diameter of fiber and an impact on the weight of the fleece. These crossbreedings with Corriedale sheep were carried out with the objective of analyzing changes on wool production, quality and skin characteristics. Evaluation parameters were fiber diameter, density of follicles and Secondary/Primary relation. Follicle count primary and secondary were made from skin biopsies and were performed with the aid of a punch. Features regarding the production and quality of wool from sheep were considered in contemporary animals, evaluating fleece weight and clean dirty, length of strands, washing yields, coefficient variation of fiber and W / D3. The comparison made in this paper would attribute the decrease in fiber diameter to the crossing with MPM. The follicle density on Corriedale ewes, Corriedale ewe lambs in and the F1 (Corriedale x MPM) were significantly different. The S / P was 9,08 ± 2,32 ; 9.46 ± 1.57 and 11,93 ± 2,18 respectively. The fleece weight in the F1 ewe lambs (Corriedale x MPM) was lower and significantly different from its Corriedale contemporaries. The yield on wash fleece at Corriedale ewe lambs was higher than ewe lambs crossbred with MPM. The differences were significant in length of strands between Corriedale and Corriedale sheep x MPM. Expected results from the crossbreeding could not be substantiated under the conditions realized. Additional studies will be needed to support possible morphological and productive investment.

Avaliação da densidade folicular em ovários de fetos humanos / Follicular density evaluation in ovaries of human fetuses

OBJETIVO: avaliar a variação da população de folículos ovarianos ao longo do desenvolvimento fetal e acrescentar dados aos escassos, incompletos e, algumas vezes, divergentes dados descritos na literatura. MÉTODOS: doze ovários de fetos necropsiados foram estudados, sendo nove de fetos e três de neonatos. As idades dos fetos foram determinadas pela cronologia e por ultra-sonografia, enquanto os neonatos nascidos na 39ª semana de gravidez faleceram nas primeiras horas de vida. As peças foram fixadas com formaldeído e incluídas em parafina. Foram realizados cortes seriados com espessura de 7 mm e a cada 50 cortes, o material foi corado com HE e analisado com microscópio com aumento de 400 vezes. Foram contados os folículos em dez diferentes regiões do córtex ovariano, cada região com uma área de 625 mm². O número total de folículos em 1 mm³ foi calculado usando-se a fórmula: Nt=(No x St x t)/do, onde Nt é o número de folículos, No é a média de folículos observados em 1 mm², St é o total de cortes em 1 mm³ do ovário, t é a espessura do corte e do é o diâmetro médio do núcleo. RESULTADOS: a idade dos fetos variou de 24 a 39 semanas. O número de folículos por 0,25 mm² variou de 10,9 ± 4,8 em um neonato até 34,7 ± 10,6 também em um neonato. Entre os fetos, tivemos o menor valor com 36 semanas (11,1 ± 6,2) e o maior valor com 28 semanas (25,3 ± 9,6). O número de cortes observados por ovário variou de seis a 13, correspondendo à contagem de folículos em áreas que variaram de 15 a 32,5 mm². O total de folículos estimado variou de 500.000, na idade de 22 semanas, a mais de 1.000.000, na idade de 39 semanas. CONCLUSÕES: nossos resultados demonstram as diferentes densidades de folículos ovarianos durante o período gestacional, contribuindo para o escasso conhecimento existente na literatura até o momento.

PURPOSE: to determine the variation of the number of ovarian follicles during fetal life. METHODS: twelve ovaries donated for research were included in our study, nine from fetuses and three from newborn babies who died in the first hour after being delivered with 39 weeks of pregnancy. Fetal age was confirmed both by the last menstrual period of the woman and by ultrasonography. Ovaries were fixed in formaldehyde, included in paraffin and serially sliced at 7 mm. At every 50 cuts, the obtained material was haematoxilin-eosin stained and evaluated with an optical microscope (400 X). The follicles were counted in ten different regions of the ovarian cortex, each region with an area of 625 mm². The presence of a nucleus was considered the parameter for counting. Follicular density, per 1 mm³ was calculated using the formula Nt=(No x St x t)/do, where Nt is the number of follicles; No is the mean number of follicles in 1 mm²; St is the total number of slices in 1 mm³; t is the slice thickness and do is the nuclei mean diameter. RESULTS: the gestational age of fetuses ranged from 24 to 39 weeks. The number of follicles per 0.25 mm² ranged from 10.9 ± 4.8 in a newborn to 34.7 ± 10.6 in another newborn. Among the fetuses, the least value was obtained in a 36 week-old fetus (11.1 ± 6.2) and the highest in a 28 week-old fetus (25.3 ± 9.6). The total number of slices per ovary ranged from six to 13, corresponding to follicles counted in areas from 15 to 32.5 mm². The total number of follicles ranged from 500,000 at the age of 22 weeks to > 1,000,000 at the age of 39 weeks. CONCLUSIONS: our results demonstrate different (increasing) densities of ovarian follicles along the gestational period, providing more knowledge about this still not well-known subject.