Quantification of multi-oocyte follicles in ovaries of bitches

ABSTRACT The objective of this study was to quantify the number and frequency of monocyte (MnOF) and multi-oocyte (MtOF) follicles in ovaries of bitches subjected to ovary salpingohysterectomy (OSH). Right and left ovaries of 38 bitches were collected after OSH, prepared, and a histological analysis was carried out. The ovaries were subjected to surface and deep histological cuts; the follicles were classified, and the number of follicles and cumulus oophorus complexes (COC) per follicle were quantified for each histological cut. MnOF and MtOF were found in all ovaries, at different developmental stages; primary follicles were grouped in the ovarian cortex, and follicles at other follicular stages presented a random distribution. MtOF containing two, three, four, or more COC were found in the ovaries of bitches, with a decreasing frequency trend, according to the number of COC in the MtOF. The effect of the age, number of estrus, estrus interval, and number of progenies per delivery was not significant for the number and frequency of MtOF in the ovaries of the bitches, whereas the size, number of pregnancies, use and number of contraceptive applications had some effect on the number and frequency of MtOF in the ovaries of the bitches.

Comparing rLH with hMG in embryo transfers at the stage of blastocyst and pregnancy outcomes in poor responders

Despite showing the beneficial effects of adding LH activity to FSH, based on the pregnancy rate (PR) in patients in the previous studies, no studies have been done to compare two different gonadotrophin preparations with activity of LH in the same group of poor responders. The present study was a single-center retrospective one conducted in January 2015 - December 2019 among 30 women under 39 years old who had reduced ovarian reserve and underwent ICSI cycles. The same patient group received rFSH combined with hMG or rLH. The ovarian stimulation cycle began on the second day of the menstrual cycle, and the initial doses of gonadotrophin were 225 IU/day rFSH in addition to 75 IU/day hMG or 75 IU/day rLH. In all cycles, a flexible antagonist protocol was used. Adding rLH significantly increased the mean number of MII oocytes and cumulus oophorus complex (COC) (p < 0.001). There was no significant difference between poor responders treated with rLH or with hMG in terms of blastocyst transfer, implantation rates and clinical pregnancy rates (p>0.05). There should be further studies to confirm the better effect of rLH addition to rFSH than hMG in young poor responders. Interventions for poor responders obviously need large, randomized studies which were designed properly, due to the lack of evidence-based treatment to date for this particular patient group.

Un sistema de co-cultivo con células del cumulus oophurus mejora la calidad de embriones bovinos producidos in vitro

El estudio tuvo como objetivo determinar la influencia de un sistema de co-cultivo con células del cumulus oophorus sobre la calidad de embriones bovinos producidos in vitro en función a su viabilidad. Se utilizaron ovarios de bovinos hembra cruzadas, sacrificadas en el Centro de Beneficio Municipal de Chachapoyas, Amazonas, Perú. Los ovocitos aspirados de folículos de 2-6 mm fueron madurados en medio TCM-199 por 24 horas, en una atmósfera humidificada con 6% de CO2 y a 38.5 °C. El estado de maduración fue determinado mediante el estadio nuclear a través de tinción aceto-orceína. Los ovocitos maduros se fertilizaron in vitro con semen congelado de toros Angus durante 18 horas. Los presuntos cigotos fueron distribuidos aleatoriamente para su desarrollo en un grupo en medio con co-cultivo y otro en medio sin co-cultivo (control). Se determinó la calidad embrionaria en función a la actividad enzimática y daño al ADN de los embriones al séptimo día del cultivo, mediante los fluorocromos diacetato de fluoresceína (FDA) y yoduro de propidio (PI), respectivamente. No se encontró asociación significativa (p>0.05) en la cantidad de embriones producidos in vitro, pero hubo diferencia significativa en viabilidad embrionaria a favor del sistema con co-cultivo (p<0.05). El uso de co-cultivos con células de cumulus oophurus mejora la viabilidad y, por lo tanto, la calidad embrionaria, sin influir en la tasa de producción de embriones in vitro.

New markers of human cumulus oophorus cells cultured in vitro – transcriptomic profile

The presence of CCs around the oocyte after ovulation is one of the key elements contributing to oocyte developmental competence. In the presented study, we used CCs from 12 patients aged 18-40 diagnosed with infertility. After harvesting cells on day 1, 7, 15 and 30 of culture, total RNA was isolated and transcriptomic analysis was performed. The DAVID software indicated the following GO BP terms: “cell junction organization”, “cell migration”, “cell morphogenesis involved in differentiation”, “cell morphogenesis” and “cell motility”. Of the genes belonging to all ontological groups, the most downregulated were: SLC7A8, DFNB31, COL1A1, CDC42SE1, TGFBR3, HMGB1, with the most upregulated genes being: ANXA3, KIAA1199, HTR2B, VCAM1, DKK1.While many studies focus on attempts to obtain fully competent oocytes, scientists still have difficulty attaining adequate results in vitro. Lack of adequate knowledge often results in low in vitro fertilization efficiency. Therefore, our research focuses on CCs cells, thanks to which the oocyte most likely acquires developmental competence. The main purpose of the study was to identify the potential molecular markers responsible for cell junction organization, migration, differentiation, morphogenesis and motility.Running title: New markers of human cumulus oophorus cells cultured in vitro

AVALIAÇÃO MORFOMÉTRICA OVARIANA E RECUPERAÇÃO DE COMPLEXOS Cumulus oophorus DE FÊMEAS SUÍNAS PRÉ-PÚBERES POR ASPIRAÇÃO FOLICULAR E SECÇÃO DOS OVÁRIOS

Os objetivos do estudo foram caracterizar as dimensões ovarianas eavaliar duas técnicas de recuperação de CCOs (complexos Cumulus oophorus) de ovários de fêmeas suínas pré-púberes, com idade média de cinco meses e peso corporal médio de 80 kg. Os ovários derivados de abatedouro (n=127) foram pesados usando uma balança digital e medidos (comprimento, largura e altura) com um paquímetro. Dos 127 ovários, 38 foram submetidos à aspiração folicular e 20 foram seccionados utilizando-se uma lâmina de bisturi. Os CCOs dos 58 ovários foram classificados em graus 1, 2, 3 e 4, com o auxílio de um estereomicroscópio. As médias de CCOs recuperados segundo a técnica foram comparadas pelo teste t (P<0,05). As variáveis foram correlacionadas pelo teste de Correlação de Pearson.Os resultados médios observados foram: peso dos ovários 4,26 g; comprimento 2,57 cm; largura 1,99 cm e altura 1,39 cm. No total, foram recuperados 5.871 CCOs dos 58 ovários. As porcentagens de cada grau foram as seguintes: 0,87%; 2,23%; 14,50% e 82,40% (graus 1, 2, 3 e 4, respectivamente). Do número total de CCOs colhidos, o grau 4 foi o mais prevalente e significativo para este experimento. O peso corporal e a largura ovariana exibem maior influência na quantidade e na qualidade dos CCOs. Conclui-se que a técnica de secção provê maior recuperação de CCOs. Quando a técnica de recuperação de CCOs é utilizada em fêmeas suínaspré-púberes prevalece a recuperação de CCOs grau 4.

Histological description of aceh cattle ovary cryopreserved by various cryoprotectants

This research aimed to determine Aceh cattle ovarian follicle morphological integrity after vitrified by various cryoprotectants. Cryoprotectants used in this research were 30% ethylene glycol (EG), 30% dimethyl suphocide (DMSO), and combination of 15% EG + 15% DMSO. Prior to vitrification process, ovaries were cleansed by phosphate buffered saline (PBS) and then cut into ±1 mm³. Ovaries were consecutively submerged into the following liquid for 5 minutes each: PBS+ 0.25 M sucrose; PBS+ 0.5 M sucrose; PBS+ 0.5 M sucrose + 10% cryoprotectants; and PBS+ 0.5 M sucrose + 30% cryoprotectants. Then, ovaries were packed into straws with ±7 cm in length and ± 6 mm in diameter. Before kept in liquid nitrogen, ovaries were first exposed to nitrogen fume for 10 second. After being stored for 1 day, the ovaries were proceed for histological examination. The result showed that Aceh cattle ovarian follicle after vitrification using 30% EG yields the best morphological integrity. Cumulus oophorus, zona pellucida, granulose cell arrangement, theca interna, and theca externa cells were observed clearer in ovary which was vitrified with 30 % EG than those with 30% DMSO and combination of 15% EG + 15% DMSO. As conclusion, 30% EG was able to protect ovary morphological integrity better than 15 % EG + 15% DMSO and 30% DMSO. Furthermore, combination of 15% EG+ 15 % DMSO was relatively better in protecting ovary follicle morphological integrity compared to 30% DMSO.