Reproductive Sciences

Magnesium sulfate is used as a tocolytic, but clinical efficacy has been seriously questioned. Our objective was to use controlled ex vivo conditions and known pregnancy stages, to investigate how 2 key factors, hormones and gestation, affect magnesium’s tocolytic ability. We hypothesized that these factors could underlie the varying clinical findings around magnesium’s efficacy. Myometrial strips were obtained from nonpregnant (n = 10), mid-pregnant (n = 12), and term-pregnant (n = 11) mouse uterus. The strips were mounted in organ baths superfused with oxygenated physiological saline at pH 7.4 and 37°C. The effect of different concentrations of MgSO₄ (2-20 mM) was examined on spontaneous and oxytocin-induced (0.5-1 nM) contractions. Contractile properties (amplitude, frequency, and area under the curve) were measured before and after application of magnesium. Magnesium sulfate had a dose-dependent inhibitory effect on both spontaneous and oxytocin-induced contractions but was less effective in the presence of oxytocin. In spontaneous contractions, magnesium was more potent as gestation progressed (P < .0001). In the presence of oxytocin, however, there were no significant gestational differences in its effects on contraction. The rapid onset and reversal of magnesium’s effects suggest an extracellular action on calcium entry. Taken together, we conclude that magnesium’s actions are influenced by both gestational state and hormones, such that, at least in mice, it is least effective in early gestation with oxytocin present and most effective at term in the absence of oxytocin. That magnesium is least effective preterm and oxytocin decreases its effectiveness throughout gestation, may explain its disappointing clinical effects as a tocolytic.

Preeclampsia (PE) is a pregnancy disorder leading to the morbidity and mortality. Despite the development of the understanding of etiology, the only effective treatment of PE is the delivery of the placenta. An improved mastery on the regulation of trophoblast invasion could be meaningful to alleviate the disease burden of PE. Relative expression of CD97 in PE and normal placental tissues was evaluated by quantitative real-time polymerase chain reaction, immunohistology, and Western blot. The CD97 siRNA and expression vector was transfected to cultured human trophoblast HTR-8/SVneo, and the cell invasion as well as the protein expression in PI3K/Akt/mTOR signaling pathway were evaluated. Expression of CD97 is significantly downregulated in PE placental tissues compared to normal controls. The Si-CD97 inhibits HTR-8/SVneo trophoblast cells invasion, as well as the activation of PI3K/Akt/mTOR signaling pathway. In accordance, overexpression of CD97 promotes trophoblast cell invasion. In addition, CD97 regulates FOXC2 expression and showed similar effects on PI3K/Akt/mTOR signaling pathway as specific FOXC2 inhibitor. In short, this study demonstrated the downregulation of CD97 expression in preeclamptic placentas. Further mechanism investigation revealed that CD97 promoted trophoblast invasion by targeting FOXC2 via PI3K/Akt/mTOR signaling pathway, laying the foundation for the development of PE intervention strategy by targeting CD97 in placentation and pathogenesis of PE.

Diminished ovarian reserve (DOR) and primary ovarian insufficiency (POI) are primary factors leading to infertility. However, there is a lack of appropriate animal models of DOR usable for assessing new therapeutic strategies. In this study, we aimed to evaluate whether chemotherapy treatment in mice could reproduce features similar of that observed in women with DOR. Twenty-one Nonobese diabetic/severe combined immunodeficiency (NOD/SCID) female mice were allocated to 3 groups (n = 7/group): control, single dose of vehicle (Dimethyl Sulfoxide [DMSO]); DOR, single reduced chemotherapy dose; and POI, single standard chemotherapy dose. After 21 days, mice underwent ovarian hyperstimulation and mating. Part of the animals were harvested to analyze ovarian reserve, ovulation and fertilization rates, and morphology, apoptosis, and vascularization of the ovarian stroma. The remaining mice underwent multiple matings to assess pregnancy rates and litter sizes. The DOR and POI mice showed an impaired estrous cyclicity and a decrease in ovarian mass, number of follicles, Metaphase II (MII) oocytes, and embryos as well as in ovarian stroma vascularization. Mice in both models showed also an increase in the percentage of morphologically abnormal follicles, stromal degeneration, and apoptosis. Similar to that observed in DOR and POI patients, these impairments were less severe in DOR than in POI mice. None of the POI females were able to achieve a pregnancy. Meanwhile, DOR females achieved several consecutive pregnancies, although litter size was decreased when compared to controls. In conclusion, a mouse model which displayed most of the ovarian characteristics and fertility outcomes of women with DOR has been established using a single dose of chemotherapy.

When the presence of endometriotic lesions are not evident by hematoxylin and eosin staining, CD10 is used to highlight and confirm the presence of endometriotic stroma. However, CD10 is not specific only to the endometrial stroma but is also expressed in many other cells. Recently, interferon-inducible transmembrane protein 1 (IFITM1) was reported as a highly specific immunohistochemical marker of normal endometrial stroma and endometrial stromal neoplasm. In this study, we examined the expression of IFITM1 and CD10 in 18 cases of ovarian endometriosis and 44 cases of extragenital endometriosis. Among the 62 patients, 62 (100.0%) were positive for IFITM1 and 60 (96.8%) for CD10, and CD10 was negative in 2 cases that were positive for IFITM1. Additionally, we found that IFITM1 sensitivity was unaffected by the presence or absence of hormonal therapy. To the best of our knowledge, this represents the first demonstration of IFITM1 as a highly sensitive stromal marker of ovarian and extragenital endometriosis.

In cases of extragenital endometriosis or microscopic endometriosis lesions, pathological diagnosis can be challenging because endometriotic stroma and glands represent only a minor component of fibrotic endometriotic lesions. For better accuracy of diagnosis, the development of a sensitive and specific epithelial marker is beneficial. Previous studies showed that PAX8 is a highly sensitive and specific marker for primary and metastatic Mullerian epithelial tumors. Therefore, we sought to examine whether PAX8 is a highly sensitive marker for glands in extragenital endometriosis. Eight and 47 samples of ovarian endometrioma and extragenital endometriosis, respectively, were evaluated in this study. We calculated the percentage of samples positively immunostained for PAX8, CD10, estrogen receptor (ER), and progesterone receptor (PR). PAX8 was positive for endometriotic epithelial cells in 95.7% (45/47) of extragenital endometrioses and in 100% (8/8) of ovarian endometrioses. CD10 was positive for endometriotic stromal cells in 97.9% (46/47) of extragenital endometrioses. PAX8 was strongly positive for glands, even in a CD10-negative case. The expression of PAX8, CD10, and PR was not affected by preoperative hormonal therapy, and the positive rate of ER staining was significantly reduced by preoperative hormonal therapy. In conclusion, PAX8 is a highly sensitive epithelial marker for extragenital endometriosis. This specific expression was maintained under hormonal therapy. It is noteworthy that extragenital endometriosis maintains the expression of this lineage marker, although it occurs at various sites, and its cause and mechanism of development might be different. PAX8 nuclear expression can be useful in detecting extragenital endometriosis in clinical practice.

Oxidative stress is one of the most detrimental factors that affect oocyte developmental competence and embryo development in vitro. The impact of anethole supplementation to in vitro maturation (IVM) media on oocyte maturation and further bovine in vitro embryo production was investigated. Oocytes of slaughterhouse-derived bovine ovaries were placed in IVM with anethole at different concentrations of 30 (AN30), 300 (AN300), and 2000 μg/mL (AN2000), or without (control treatment). The oocytes were assessed for maturation rates, and for reactive oxygen species (ROS) and ferric reducing antioxidant power (FRAP) levels, and mitochondrial membrane potential. Embryo development was assessed by cleavage and blastocyst rates, and embryo cell number. The percentage of metaphase II oocytes were similar among the treatments (range, 77%-96%). Anethole at 300 µg/mL was the only treatment that yielded higher cleavage and embryo development (morula and blastocyst) rates compared to the control treatment. The ROS production in the oocytes after maturation did not differ among treatments. However, oocytes treated with anethole at 300 µg/mL had higher (P < .05) FRAP and mitochondrial membrane potential compared to the control treatment. Furthermore, AN300 treatment increased (P < .05) the average number of total cells in blastocysts compared to the control and AN30 treatments. The use of anethole at 300 μg/mL during IVM is suggested to improve the quantity and quality of bovine embryos produced in vitro. The beneficial effects of anethole on embryonic developmental competence in vitro seems to be related to its capacity to regulate the redox balance and improve mitochondrial function in oocytes and embryos.

Abnormal expression of SPARC (osteonectin), cwcv and kazal-like domains proteoglycan 2 (SPOCK2) plays a significant role in the development and progression of various human cancers, yet a relationship between SPOCK2 and endometrial cancer (EC) has not been reported. Here, we assessed the potential role and mechanism by which SPOCK2 acts in the pathogenesis and progression of EC. First, protein expression of SPOCK2 in EC tissue from patients was detected by immunohistochemistry and associated clinical data were analyzed. Then, HEC-1A and Ishikawa cells were transfected with an adenoviral vector containing an SPOCK2 recombinant fragment and the biological behavior of transfected cells was observed. Finally, the expression of membrane type 1 matrix metalloproteinase (MT1-MMP) and MMP2 in the transfected cells was detected by Western blot and zymography gel assay to analyze the effect of SPOCK2 on the regulation of the MT1-MMP/MMP2 pathway. We found that there was significantly less SPOCK2 protein expression in the EC tissue than in the normal endometrium tissue, and lack of SPOCK2 protein expression in EC tissue was associated with distant metastasis and myometrial invasion. Upregulation of SPOCK2 in HEC-1A and Ishikawa cells inhibited cell proliferation, invasion, adhesion, and apoptosis. Upregulation of SPOCK2 inhibited the expression of MT1-MMP and MMP2 and activation of MMP2 in HEC-1A and Ishikawa cells. Collectively, our data indicated that SPOCK2 contributed to the progression of EC by regulating the biological behavior of cancer cells, which is achieved partly through regulating protein expression of MT1-MMP and MMP2 and activation of MMP2.

Preterm birth is a leading cause of neonatal mortality in the US and globally, with preterm premature rupture of fetal membranes (PPROM) accounting for one third of preterm births. Currently no predictive diagnostics are available to precisely assess risk and potentially reduce the incidence of PPROM. Bigycan and decorin, the main proteoglycans present in human fetal membranes, are involved in the physiological maturation of fetal membranes as well as in the pathophysiology of preterm birth. The serum protein sex hormone-binding globulin (SHBG) has recently been identified as a predictor of spontaneous preterm birth. We hypothesize that the balance between serum decorin and biglycan on one hand and SHBG on the other hand may provide insight into the status of the fetal membranes in early pregnancy, thereby predicting PPROM prior to symptoms. Using chart review, 18 patients with confirmed cases of PPROM were identified from 2013-2016. Second trimester residual serum was retreived from freezer storage for these cases along with 5 matched controls for each case. The biomarkers biglycan, decorin and SHBG were analyzed first separately, then in combination to determine their ability to predict PPROM. The predictive score for the combined model displays an AUC = 0.774. The ROC curve of the predicted score has an optimal threshold of 0.238 and a sensitivity and specificity of 0.72 and 0.84 respectively. This prenatal serum panel is a promising serum screening-based biochemical model to predict PPROM in asymptomatic women.

Thoracic endometriosis (TE) syndrome is a clinical condition known as an extrapelvic form of endometriosis with the presence of functioning endometrial tissue involving lung parenchyma, pleura, chest wall, or diaphragm. In an effort to obtain an endometriosis ex vivo model, we established the spontaneously growing TH-EM1 cell line from endometriotic implants in lung parenchyma from a woman with TE. Maintained in long-term culture, the cells grew as large mesenchymal-like cells with a doubling time between 5 and 6 days. Treatment with medroxyprogesterone acetate (10⁻⁷ mol/L) inhibited the TH-EM1 cells growth and induced morphological changes to an epithelial-like cells. Strong expression of the nuclear estrogen receptors, progesterone receptors, and erytropoietin receptors were found in both the pulmonary implant and the TH-EM1 cells by immunohistochemical analysis. Consistent immunoreactivity of TH-EM1 cells for CD9, CD13, CD73, CD90, CD105, and CD157 was revealed by flow cytometry. Likewise, the embryonic markers, SRY-box 2 (SOX-2) and the Nanog molecules, were detected in 76% and 52% of the cells, while fetal hemoglobin and α-globin were detected in 76% and 65% of TH-EM1 cells, respectively. By RHG banding, normal metaphases were observed, while the microarray chromosomal analysis showed gains of DNA sequences located on the segments 8p23.1, 11p15.5, and 12p11.23. The described in vitro cellular model can serve as a useful tool to study the pathogenesis of endometriosis and to improve the knowledge of molecular mechanisms controlling the endometriotic cell dissemination potential.

Indoleamine 2, 3-dioxygenase (IDO), an immunosuppressive enzyme that mediates the conversion of tryptophan to kynurenine, was shown to play a key role in placental development during normal pregnancy. However, little is known about the pattern of IDO expression in the endometrium and its attendant functional significance in pregnancies complicated with recurrent miscarriage (RM). Immunohistochemical studies of IDO, Foxp3, CD56, and CD163 expression were performed in endometrial samples from women with RM and healthy fertile controls. Our study found that IDO was localized in glandular epithelial cells, surface epithelial cells, and a small number of cells within the stromal compartment (including stromal cells and leukocytes) in endometrium. Indoleamine 2, 3-dioxygenase expression in the RM group was significantly lower than control group. The Foxp3 and CD56 expression were significantly increased with the elevated IDO expression in controls but not in RM. The percentage of Foxp3+ Tregs was significantly correlated with the level of IDO expression in the control group. Comparatively, no correlation was found between the percentage of CD56+ cells, CD163+ cells, and the level of IDO expression, no matter in controls and RM patients. This study demonstrated that the downregulation of IDO expression and noncoordinated association between IDO and other endometrial immune cells were associated with RM. Our findings provide insights into the contribution of IDO in immune regulation to maintain normal pregnancy, which could be used to develop potential therapeutic methods for RM.

Recurrent pregnancy loss (RPL) affects up to 5% of couples. It is believed that genetic factors contribute to the disease’s etiology and pathophysiology. Hundreds of genes represent coherent RPL candidates due to mammalian implantation’s inherent complexity. Sanger sequencing (direct sequencing) of candidate genes has identified potential RPL causative genes (and variants), including those regulating embryo implantation and pregnancy maintenance. Although this approach is a reliable technique, the simultaneous analysis of large genomic regions is challenging. Next-generation sequencing (NGS) technology has thus emerged as a useful alternative for determining genetic variants and transcriptomic disturbances contributing to monogenic and polygenic diseases pathogenesis. However, interpreting results remains challenging as NGS experiments provide an enormous amount of complex data. The molecular aspects of specific diseases must be fully understood for accurate interpretation of NGS data. This review was thus aimed at describing (for the first time) the most relevant studies involving Sanger and NGS sequencing, leading to the description of variants related to RPL pathogenesis. Successful RPL-related NGS initiatives (including RNAseq-based studies) and future challenges are discussed. We consider that the information given here should be useful for clinicians, scientists, and students to enable a better understanding of RPL etiology. It may also provide a basis for the development of diagnostic/prognostic approaches contributing toward translational medicine.

MicroRNAs (miRNAs) are a novel class of small noncoding RNAs (ncRNAs) that play critical roles in regulation of gene expression, especially at posttranscriptional level. Over the past decade, the degree to which miRNAs are involved in male infertility has become clear. They are expressed in a cell- or phase-specific manner during spermatogenesis and play crucial role in male reproductive health. Therefore, dysregulation of miRNAs in testicular cells can be considered as a molecular basis for reproductive failure and male infertility. The abnormal expression pattern of miRNAs can be transmitted to the offspring via assisted reproductive techniques (ART) and results in the birth of children with a higher risk of infertility, congenital abnormalities, and morbidity. This review expounds on the miRNAs reported to play essential roles in somatic cells development, germ cells differentiation, steroidogenesis, normal spermatogenesis, sperm maturation, and male infertility, as well as emphasizes their importance as minimally invasive biomarkers of male infertility.

Chemokines and chemokine receptors have been implicated a pivotal role in trophoblast differentiation and in the formation of a functionally normal placenta. In this study, we present data that highlight the involvement of chemokine ligand 17/chemokine receptor 4 (CCL17/CCR4) expression at the fetomaternal interface and expand its biological relevance of influence during trophoblast differentiation and placentation. By immunohistochemistry, we found that CCL17 was abundantly expressed in the decidua and trophoblasts, especially in cell columns. The receptor for CCL17, CCR4, was specifically expressed in invading interstitial extravillous trophoblasts. Furthermore, by transwell migration, invasion assays, and the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, we found that exogenous CCL17 concentrations paralleled the invasive potential of cytotrophoblasts to some extent, with no significant effect on cell proliferation. Using Western blotting, we demonstrated that the stimulatory effect of CCL17 was related to the expression of matrix metalloproteinase 2 (MMP-2), MMP-13, integrin α5, and integrin β1, although it downregulated tissue inhibitors of MMP-1 expression. In conclusion, our findings suggest that CCL17, as a differentiation-related molecule coexpressed by decidua and trophoblast, stimulates extravillous trophoblast migration and directs invasion mainly via modulating integrins, MMPs, and its inhibitor levels.