Reproductive and Developmental Medicine

Luteotropic roles of glucocorticoids in rat granulosa cells Xin-Yan Huang, Yin-Yan Xu, Juan Xie, Li Wang Reproductive and Developmental Medicine 2020 4(4):195-203 Objective: The study objective was to investigate whether endogenous glucocorticoids directly impact the functions and proliferation/apoptosis of ovarian granulosa cells. Methods: Primary rat ovarian granulosa cells were cultured and treated with graded concentrations of corticosterone either alone or in the presence of the indicated drugs. After 48 h of treatment, the cells and growth media were collected to measure intracellular and extracellular progesterone/estradiol concentrations, and steroid secretion ratios were obtained by parameter calculation. The number of granulosa cells was determined by Cell Counting Kit-8. To determine the impact on cell numbers, granulosa cell proliferation was detected using the BrdU incorporation method and cell apoptosis was detected by flow cytometry. Results: First, high corticosterone concentrations significantly stimulated progesterone synthesis/secretion and inhibited estradiol synthesis in cultured granulosa cells. Second, accompanied by follicle-stimulating hormone, high corticosterone concentrations promoted progesterone synthesis/release and estradiol release. Last, high corticosterone concentrations increased the cell number and suppressed apoptosis but did not induce cell proliferation. Conclusions: These indicate that high glucocorticoid concentrations may play luteotropic roles in the functions and number of corpora lutea.

Benzyl alcohol-benzyl benzoate clearing reveals the dose-dependent effect of cyclophosphamide on follicle damage in mice Qi-Wang Lin, He Fei, Yun-Feng Jin, Kun-Peng Wu, Xin Dai, Ying Qu, Hua Jiang Reproductive and Developmental Medicine 2020 4(4):204-211 Objective: Cyclophosphamide (CTX), which is commonly used in clinical chemotherapy, has a damaging effect on ovarian follicles. This study aimed to establish a new method to count the number of follicles in mouse ovaries using benzyl alcohol–benzyl benzoate (BABB)-based tissue-clearing technology and evaluate the follicle-damaging effects of different doses of CTX. Methods: C57BL/6 mice were divided into four groups and administered intraperitoneal injections of 0, 40, 80, or 120 mg/kg CTX. The serum levels of estradiol (E2) and follicle-stimulating hormone (FSH) were detected using an ELISA kit. Mouse ovaries were subjected to BABB clearing and labeled with DAPI, β-actin, and DDX4 to observe the ovarian structure and follicles. A three-dimensional software, Imaris, was used to reconstruct the ovarian structure and automatically count the number of follicles. The effects of different CTX doses on the total follicle number and estrous cycle were determined. Results: As the CTX dose increased, E2 levels in CTX mice declined from 212.3 to 57.7 pg/mL; the FSH levels increased from 3.2 to 29 ng/mL. Mouse ovaries became transparent after BABB treatment. After fixation, microscopy, and Imaris processing, immunofluorescence signals of β-actin and DAPI from all levels in intact ovaries could be obtained and follicle number in half ovaries could be automatically counted using anti-DDX4 antibody labeling. In the NC, CTX40, CTX80, and CTX120 groups, the proportion of mice in the diestrus phase was 26.67%, 51.67%, 73.33%, and 95.00%, respectively, and the total follicle number was 2,603, 1,761, 1,043, and 262, respectively. E2 levels were positively correlated with follicle number and FSH levels were negatively correlated with follicle number, indicating that the damaging effect of CTX on follicle number may be dose dependent. Conclusion: BABB can be used to clear intact ovaries from adult mice, and follicle number in half ovaries can be automatically counted. The damaging effect of CTX on follicles and the endocrine system is dose dependent.

Selection and evaluation of optimal reference genes for quantitative reverse transcription-polymerase chain reaction analyses of gene expression in human spermatozoa Chun-Hai Luo, Yun-Ge Tang, Shi-Hao Hong, Yuan Tang, Ying Zhang, Fei Sun Reproductive and Developmental Medicine 2020 4(4):212-218 Objective: Optimal reference genes are critical for accurate normalization and reliable interpretation of gene expression quantification data. Recently, several strategies have been utilized for validating reference genes in different human tissues. However, no universal reference genes have been described that accurately summarize transcriptional activity in human spermatozoa. Methods: Using quantitative reverse transcription-polymerase chain reaction (RT-qPCR), we evaluated ten commonly used candidate reference genes between two groups of human cryopreserved donor sperm with different pregnancy rates. We assessed the stability of reference genes using three different algorithms, namely geNorm, NormFinder, and BestKeeper. We then identified the most stable reference genes. Results: Male-enhanced antigen 1 (MEA1) was identified as the most stably expressed reference gene, followed by testis-enhanced gene transcript (TEGT). Conclusions: We comprehensively identified MEA1 and TEGT as the most stably expressed reference genes for the normalization of gene expression data in human spermatozoa.

Alteration of apoptotic gene expression and reproductive parameters through Thymus vulgaris administration following paclitaxel-induced testopathy in micea Shiva Roshankhah, GholamReza Hassanzadeh, Azita Faramarzi, Mohammad Reza Salahshoor Reproductive and Developmental Medicine 2020 4(4):219-227 Objective: Paclitaxel (PAC) is a chemotherapy drug and has an active role in the treatment of lung, breast, and ovarian cancers; however, its use causes increased levels of free radicals, leading to severe and irreversible organ damage. Thymus vulgaris is a species of flowering plant that contains various antioxidant chemical compounds. This study aimed to investigate the effect of T. vulgaris on PAC-induced oxidative damage in mice testis and sperm parameters. Methods: In this study, 40 BALB/c mice were divided into five groups: normal control (NC); positive control (20 mg/kg of PAC); and three treatment groups (4.5, 9, and 18 mg/kg doses of T. vulgaris extract + 20 mg/kg PAC). Treatments were administered intraperitoneally daily for 50 days. Finally, the levels of serum luteinizing hormone (LH), follicle-stimulating hormone (FSH), and testosterone were measured using immunoassay method. Testicular stereological features and sperm parameters were also calculated. Antioxidant parameters were measured using nitrite oxide (NO) and perioxidation levels and ferric-reducing ability of plasma assay. The expressions of p53, caspase-3, Bax, and Bcl-2 were measured through real-time quantitative polymerase chain reaction. Results: T. vulgaris + PAC treatments at all doses significantly increased all parameters in NC and three treatment groups compared with the positive control group (20 mg/kg of PAC) (P < 0.05), except the LH, FSH, and NO levels, which were decreased. Further, significantly downregulated levels of p53, caspase-3, and Bax genes and? upregulated levels of Bcl-2 gene expression were noted in NC and three treatment groups in comparison with the positive control group (P < 0.05). Conclusions: T. vulgaris administration attenuates the toxic effects of PAC on male reproductive parameters.

Potential application of anti-müllerian hormone in polycystic ovary syndrome according to chinese classification criteria: A retrospective analysis Ling-Li Tang, Ling-Shan Zhang, Xiao-Yong Zhu, Ying-Li Shi Reproductive and Developmental Medicine 2020 4(4):228-232 Objective: Anti-Müllerian hormone (AMH) expression is elevated in patients with polycystic ovary syndrome (PCOS), however, its clinical significance is not clear. Owing to the strong correlation between AMH and polycystic ovarian morphology (PCOM), some studies believe that AMH alone can be used to diagnose PCOS. The aim of this study was to explore whether AMH can be used to diagnose PCOS and to differentiate the various PCOS subtypes. Methods: This was a retrospective study of 503 patients with PCOS. Patients were divided into eight subtypes based on the presence/absence of hyperandrogenemia (HA), insulin resistance (IR), or obesity (OB). The expression characteristics of AMH in each subtype were analyzed. Due to the small number of patients with subtypes 7 and 8, only patients with subtypes 1–6 were included in the analysis. Results: AMH showed a good positive correlation with PCOM (P = 0.000) and negative correlations with OB (P = 0.000) and IR (P = 0.003). The free testosterone index showed no correlation with AMH (P = 0.803). The percentages of patients with each subtype (excluding subtypes 7–8) and their respective AMH levels were as follows: Type 1 (HA + NIR + OB) 4.77% and 9.12 ng/mL; Type 2 (HA + IR + NOB) 20.68% and 10.34 ng/mL; Type 3 (HA + NIR + NOB) 23.66% and 9.47 ng/mL; Type 4 (HA + IR + OB) 30.82% and 8.32 ng/mL; Type 5 (NHA + NIR + NOB) 11.73% and 10.0 ng/mL; and Type 6 (NHA + IR + NOB) 6.16% and 9.76 ng/mL. The diagnostic rates of AMH (>8.09 ng/mL) and ultrasound for PCOM were 60.10% and 85.60%, respectively, suggesting that AMH did not completely predict PCOM. Conclusions: High AMH levels can be used to evaluate the incidence trend of PCOS. However, due to clinical heterogeneity, accurately evaluating the severity of PCOS and identifying the subtype of PCOS in Chinese patients are difficult. Individualized treatment should be administered based on accurate clinical subtypes and other clinical characteristics.

Prediction of cumulative live birth rate in women aged 40 years and over undergoing In vitro fertilization/intracytoplasmic sperm injectiona Lei Jia, Pan-Yu Chen, Ying-Chun Guo, Zhi-Qiang Zhang, Xiao Gong, Jing-Bo Chen, Cong Fang Reproductive and Developmental Medicine 2020 4(4):233-238 Objective: To investigate cumulative live birth rate (cLBR) per oocyte retrieval in infertile patients aged 40 years and over undergoing their first in vitro fertilization/intracytoplasmic sperm injection cycles and to identify the possible predictors. Methods: A total of 1,613 patients at a university hospital in China from January 2013 to May 2017 were enrolled in this retrospective study. All data for fresh and subsequent frozen-thawed cycles were analyzed. Multivariate logistic regression analysis with stepwise selection of possible predictors for cLBR was performed, and Loess curve was constructed to determine the association between cLBR and the number of oocytes retrieved. Results: cLBR significantly increased with the number of oocytes retrieved and reached up to 75% when > 20 oocytes were retrieved ( P < 0.001). Variables of antral follicle count (AFC) and the number of oocytes retrieved were selected using multiple logistic regression analysis with stepwise selection to predict the significance of cLBR. cLBR demonstrated an obvious upward trend as the number of oocytes retrieval increased in the Loess curve. Conclusions: For patients aged 40 years and over, AFC and the number of oocytes retrieved were two key predictors for cLBR and maximization of ovarian reserve exploitation was pivotal to increase the chance of live birth.

Consensus on infertility management and fertility preservation related to polycystic ovary syndrome Expert Group of Consensus on Infertility Managemen and Fertility Preservation Related to Polycystic Ovary Syndrome Reproductive and Developmental Medicine 2020 4(4):239-248 There is currently no universally accepted unified diagnostic standard for infertility treatment and maternity protection related to polycystic ovary syndrome (PCOS) worldwide, and the treatment options vary greatly. To improve the understanding of PCOS-related infertility among Chinese obstetricians and gynecologists at all levels for better patient diagnosis and management, we have referred to the international expert recommendations and literature and taken into account the Chinese clinical practice to develop the “Consensus on infertility management and fertility preservation related to PCOS.”

Toll-like receptor-dependent antiviral responses at the maternal–fetal interface Xiao-Rui Liu, Xiao-Wei Wei, Yi Lin Reproductive and Developmental Medicine 2020 4(4):249-256 The maternal–fetal interface is a key barrier to protect the fetus from infection. Toll-like receptors (TLRs) at the maternal–fetal interface are involved in antiviral responses. TLRs are expressed in both maternal decidua and fetal trophoblasts. Virus-induced activation of TLR signaling pathways triggers the release of interferon-related antiviral molecules and other inflammatory cytokines and/or chemokines by the host innate immune system, which may disrupt immune tolerance at the maternal–fetal interface and lead to pregnancy complications. In this review, we summarize the state of knowledge on the most common viral infections during pregnancy, antiviral TLR responses at the maternal–fetal interface, and TLR-associated pregnancy complications.

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Medicine by Alexandros G. Sfakianakis,Anapafseos 5 Agios Nikolaos 72100 Crete Greece,00302841026182,00306932607174,alsfakia@gmail.com,

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