International Journal of

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Background: CatSper genes are a novel family of four sperm-specific calcium channels, which indicate testis-specific expression patterns. Despite the crucial role of CatSper genes in the male reproduction, very little is known about the factors that regulate their expression.
Objective: The objective of this study was to investigate the effects of vitamin E treatment on the expression of CatSper 1 and CatSper 2 genes as well as sperm quality in the aged male mice.
Materials and Methods: Twenty four 11-12 months old aged male mice and twenty four 2-3-months old young male mice were randomly divided into four groups. Control groups received no injection. The experimental groups of male mice were received intraperitoneal injection of 106 mg/kg vitamin E daily for 35 days. Left testis and cauda epididymides from each mouse were collected on the days 21, 28 and 35 following vitamin E treatment and were used for Real-Time PCR and immunohistochemistry. Also, sperm analysis was performed according to the WHO guidelines given for human sperm examination. Data were analyzed using SPSS software.
Results: Administration of vitamin E improved sperm parameters in the aged as well as young adult male mice. In addition, the expression of CatSper genes increased following vitamin E treatment. Also, intensity of signal for CatSper1 and CatSper2 increased in the head and middle piece of sperm in experimental group as compared to those of control ones.
Conclusion: The vitamin E treatment significantly improved the sperm quality, especially in terms of sperm motility, count and morphology rate. Furthermore, CatSper genes expression could be up-regulated by the vitamin E treatment.

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Background: Aging contains morphological and functional deterioration in biological systems. D-galactose (D-gal) generates free radicals and accelerates aging. Portulaca oleracea (Purslane) may have protective effect against oxidative stress.
Objective: Purslane ethanolic extract effects were evaluated on antioxidant indices and sex hormone in D-gal aging female mice.
Materials and Methods: 48 female NMRI mice (25-35 gr) were randomly divided into, 6 groups: 1- control (normal saline for 45 days), 2- Purslane (200 mg/kg for last 3 weeks), 3-D-gal (500 mg/kg for 45 days), 4-D-gal+Purslane, 5- Aging, 6-Aging+Purslane. Sex hormones, antioxidants and malondialdehyde (MDA) level of ovary and uterus were measured. Histological assessment was also done.
Results: In D-gal treated and aging animals, LH and FSH levels were significantly increased (p<0.001) while estrogen and progesterone levels were significantly reduced (p<0.001) in comparison with control group. MDA contents were significantly increased in ovaries and uterus of D-gal and aging groups (p<0.01). Superoxide dismutase (SOD) (p<0.001) and catalase (p<0.01) activities were significantly decreased in both aging and D-gal treated animals. Ovarian follicles were degenerated and atrophy on uterine wall and endometrial glands was observed in D-gal and aging groups. Alteration in hormone levels, MDA contents and antioxidant activity were significantly reversed by Purslane (p<0.05). Purslane could also improve histological changes such as atrophy of endometrium.
Conclusion: These findings indicate that Purslane can attenuate aging alternations induced by D-gal and aging in female reproductive system.

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Background: Placenta adhesive disorder (PAD) is one of the most common causes of postpartum hemorrhage and peripartum hysterectomy. The main risk factors are placenta previa and prior uterine surgery such as cesarean section. Diagnosis of placenta adhesive disorders can lead to a decrease of maternal mortality and morbidities.
Objective: The purpose of this study was to compare the accuracy of color Doppler ultrasonography and magnetic resonance imaging (MRI) in the diagnosis of PADs.
Materials and Methods:In this is cross-sectional study, Eighty-two pregnant women who were high risk for PAD underwent color Doppler ultrasound and MRI after 18 weeks of gestation. The sonographic and MRI findings were compared with the final pathologic or clinical findings. P<0.05 was considered statistically significant.
Results: Mean maternal age was 31.42±4.2 years. The average gravidity was third pregnancy. 46% of patients had placenta previa. The history of the previous cesarean section was seen in 79 cases (96%). The diagnosis of placenta adhesive disorder was found in 17 cases (21%). Doppler sonography sensitivity was 87% and MRI sensitivity was 76% (p=0.37). Doppler sonography specificity was 63% and MRI specificity was 83% (p=0.01).
Conclusion: Women with high-risk factors for PAD should undergo Doppler ultrasonography at first. When results on Doppler sonography are equivocal for PAD, MRI can be performed due to its high specificity.

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Background: Perineural cysts formed within the nerve-root sheath at the dorsal root ganglion. They are most commonly located in the arachnoid covering the junction of the dorsal ganglion and nerve root. They are usually asymptomatic, incidental findings, usually located in the lumbar and sacral region of the spinal canal. It is important to consolidate the imaging findings of this rare disease so clinicians can become more clinically relevant in the evaluation of these cysts.
Case: Herein we report a case of perineural cyst misdiagnosed with hydrosalpinx by pelvic ultrasonography and finally diagnosed with magnetic resonance image.
Conclusion: Perineural cyst should be considered, in the presence of bilateral adnexal masses separated from the ovaries in pelvic sonography.

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Background: The quality of oocyte is often considered as a limiting factor for fertility, especially IVF. Some mitochondrial mutations, particularly the 4977-bp deletion increase with the age. Thus, this mutation can serve as a marker for cell aging, which indicates the reduced quality of the oocytes for fertilization. It has been suggested that this can also be investigated in the blood cells of women with IVF failure.
Objective: 1-Determination of the frequency of 4977-bp deletion in women with IVF failure, 2-Investigation of the relationship between 4977-bp deletion and the age of patients.
Materials and Methods: Polymerase chain reaction was used to detect the 4977-bp deletion in blood samples of 52 IVF failure women and 52 women who had at least one healthy child. After polymerase chain reaction with deleted and wild-type primers, the products were examined using agarose gel electrophoresis.
Results: 48.07% of women with IVF failure and 34.62% of healthy women had a mitochondrial 4977-bp deletion, with p=0.163 and OR: 1.749. Also, in association with the age of these patients and the frequency of 4977-bp mutation, p and OR were obtained 0.163 and 1.749, respectively and frequency of this mutation was higher in patients over 35 yr old compared to other subgroups (Patients ≥35: 57.69).
Conclusion: According to the findings of this study, there is no a significant relationship between the frequency of mitochondrial 4977-bp mutation and failure in IVF.

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Abstract
Color Doppler of perifollicular vascularity is a useful assessment tool to predict the growth potential and maturity of Graafian follicles. Power Angio is independent of the angle of insonation and morphometry and provides reliable clues to predict the implantation window of the endometrium. Color Doppler can be used for the prediction of ovarian hyperstimulation syndrome. It can also be used to identify the hyper responder and gonadotropin-resistant type of polycystic ovaries. The secretory scan of corpus luteum can accurately predict its vascularity and functional status. A corpus luteum with decreased blood flow is a very sensitive and specific indicator of threatened and missed abortions. Color Doppler and Power Angio need to be standardized and identical settings should be maintained if different patients, or if changes over time within the same patient are to be compared.

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Abstract
Background: Aging is accompanied by decreasing general function in the cells and tissues. D-galactose (D-gal) induces aging and plays a role in the pathogenesis of it. Myricitrin is a plant-derived antioxidant.
Objective: The present study was performed to evaluate the effects of myricitrin on antioxidant defense, sex hormone levels, uterus, and ovarian histology in D-gal-induced aging female mouse model.
Materials and Methods: In this experimental study, 72 female adult NMRI mice, weighing 30-35 gr, 3-4 months old, were randomly divided into six groups (n = 12/each): (I) Control (vehicle; normal saline), (II) D-gal at 500 mg/kg/d for 45 days, (III-V) D-gal + myricitrin-treated groups (these groups received myricitrin at 5, 10, and 20 mg/kg/d, and (VI) D-gal + 100 mg/kg/d vitamin E orally for the last 28 days. The antioxidant indices were done on the basis of colorimetric method, and sex hormone levels were measured by using enzyme-linked immunosorbent assay kits. Histological assessment of the uterus and ovaries were also evaluated.
Results: D-gal impaired the estrous cycle, also degenerative changes occur in the ovarian follicles and damage to the uterus and ovarian tissue occurs. In D-gal group, the level of sex hormones (p =0.03) and the total antioxidant capacity (p = 0.002) decreased, while the level of malondialdehyde and gonadotropins increased (p = 0.03). Myricitrin at lower doses and vitamin E ameliorated the D-gal effects.
Conclusion: These findings suggest that myricitrin at low doses can effectively prevent D-gal-induced oxidation and aging in mice. The effect of myricitrin was equivalent and sometimes better than vitamin E.

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Background: The ovarian follicular development of rodents begins at neonatal period, the stage at which primordial follicles are formed. During estrous cycle, most of the follicles undergoes atresia and some of them continue their development process. The mammalian’s ovary is regulated by some factors including hormonal factors and direct neuron effects. Previous studies have shown that the fate of follicles in this cycle are affected by hormones such as follicle-stimulating hormone and luteinizing hormone. In addition, there are two different populations of neurons in ovary, the internal and external neurons. External nervous system of mouse ovary has many roles. Several studies have shown its role in developmental process, cyclic stages, pregnancy, and aging process. These nerves and also ganglia are responsible for ovarian estradiol secretion. Some studies implied that the ganglia in ovary takes part in some functions such as hormone secretion but to best of our knowledge, their relationship with follicular and ovarian development have not fully understood.
Objective: The present study was set out to investigate two-dimensional (2D) and three-dimensional (3D) evaluations of ovarian nervous network development and the structural relationship between folliculogenesis and gangliogenesis in mouse ovary.
Materials and Methods: Adult mice ovarian tissue samples were collected from diestrus and estrus stages. In details, firstly, the cardiac perfusion was performed. The collected ovarian samples were stained by a Golgi-Cox protocol. Following staining, tissues were serially sectioned with thickness of 30 μm for each section for imaging and further analysis. Ovarian tissue serial images were evaluated with Image J software for 2D analysis and with Imaris software for 3D analysis. The images of estrus and diestrus ovaries were separately compared. In addition, the 2D and 3D data of estrus ovary were comparably analyzed. IBM SPSS Statistics 26 software was used for statistical analysis. The mean differences between follicular groups were analyzed by one-way ANOVA and post hoc Tukey test.
Results: Neural filaments and ganglia were detected in the ovaries by Golgi-Cox staining. In both 2D and 3D studies, an increase in the number and area of ganglia was seen during the follicular growth (p < 0.05). The same pattern was also seen in corpora lutea development. However, in some cases such as ratio of ganglia number to follicle area, the ratio of ganglia area to follicular area, 2D findings were different compared with the 3D results. 3D analysis of ovarian gangliogenesis showed the possible direct effect of them on folliculogenesis. Golgi-Cox staining was used in this study for 3D evaluation in non-brain tissue. The results of 3D analysis of the present study showed that, in some cases, the information provided by 2D analysis does not match the reality of ovarian neuronal function. This confirmed the importance of 3D analysis for evaluation of ovarian function.
Conclusion: It was demonstrated that there was positive relationship between gangliogenesis and folliculogenesis in mouse ovary. Ovarian ganglia, as an independent part of ovarian nervous system, is likely to have an important role in folliculogenesis and luteogenesis. Additionally, Golgi-Cox staining and 3D tissue imaging, instead of 2D imaging, are promising protocols for study of ganglia in ovarian tissue.
Key words: 3D Imaging, Ganglia, Ovarian follicle, Golgi-Cox staining, Mice.
The original full text of this abstract has been published in scientific reports 2021; 11, 5547 (2021). https://doi.org/10.1038/s41598-021-84835-0.
How to cite to this article: Asadi Zarch M.E., Afshar A., Rahmanifar F. et al. Three-dimensional and two-dimensional relationships of gangliogenesis with folliculogenesis in mature mouse ovary: a Golgi–Cox staining approach. Scientific Reports 2021; 11: 5547.

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