Reproductive Sciences

Although inositol dietary deficiency in the general population has not been demonstrated at the serum level, several findings are emerging regarding the impact of inositol supplementation in periconceptional period and in early phases of pregnancy. We are aimed to summarize all experimental (murine in vivo and in vitro murine embryo studies) and clinical (human) evidences regarding the role of inositol in the prevention and treatment of folate-resistant embryo neural tube defects (FR-NTDs) and gestational diabetes mellitus (GDM). We also collected all information regarding the effect that inositol supplementation may have in the metabolic reassessment of early and late pregnancy in order to draw evidence-based conclusions and suggest further studies defining the potential therapeutic role of this molecule in human reproduction. The systematic review of literature clearly showed that inositol supplementation in preconceptional period and in early phase of pregnancy reduces the risk of developing GDM in patients at increased risk. Furthermore, continued intake during pregnancy improves the metabolic status of affected patients, but further studies are needed to confirm this end point. All women at risk of FR-NTDs assuming inositol from the periconceptional period until late pregnancy are reported to have healthy newborns without any significant complications linked to inositol supplementation.

The divergent requirement for tolerance to support conception and protective response against sexually transmitted infections defines the unique immunological dynamics in the female reproductive tract (FRT). In part, these requirements are achieved by the cyclic modulation of cytolytic CD8T cell function in the FRT that underlie the respective immunosuppressive and immunocompetent milieus during the secretory and proliferative phases of the menstrual cycle. The CD8T cell function can be dampened by exposure to indoleamine 2,3-dioxygenase and/or arginase enzymes. Indeed, these 2 enzymes are known as primary inducers of immune suppression in tumor microenvironments. This review discusses the intriguing parallel expression of these 2 enzymes in tumor microenvironments and in the secretory endometrium. We surmise that investigating the underlying natural mechanisms that suppress and restore the immunocompetence of CD8T cells in the FRT each month may provide valuable insights into ways to artificially recapitulate these mechanisms and inhibit immune suppression in tumor microenvironments.

Uterine leiomyoma are a common benign pelvic tumors composed of modified smooth muscle cells and a large amount of extracellular matrix (ECM). The proteoglycan composition of the leiomyoma ECM is thought to affect pathophysiology of the disease. To test this hypothesis, we examined the abundance (by immunoblotting) and expression (by quantitative real-time polymerase chain reaction) of the proteoglycans biglycan, decorin, and versican in leiomyoma and normal myometrium and determined whether expression is affected by steroid hormones and menstrual phase. Leiomyoma and normal myometrium were collected from women (n = 17) undergoing hysterectomy or myomectomy. In vitro studies were performed on immortalized leiomyoma (UtLM) and normal myometrial (hTERT-HM) cells with and without exposure to estradiol and progesterone. In leiomyoma tissue, abundance of decorin messenger RNA (mRNA) and protein were 2.6-fold and 1.4-fold lower, respectively, compared with normal myometrium. Abundance of versican mRNA was not different between matched samples, whereas versican protein was increased 1.8-fold in leiomyoma compared with myometrium. Decorin mRNA was 2.4-fold lower in secretory phase leiomyoma compared with proliferative phase tissue. In UtLM cells, progesterone decreased the abundance of decorin mRNA by 1.3-fold. Lower decorin expression in leiomyoma compared with myometrium may contribute to disease growth and progression. As decorin inhibits the activity of specific growth factors, its reduced level in the leiomyoma cell microenvironment may promote cell proliferation and ECM deposition. Our data suggest that decorin expression in leiomyoma is inhibited by progesterone, which may be a mechanism by which the ovarian steroids affect leiomyoma growth and disease progression.

Mammalian target of rapamycin (mTOR) signaling serves as a central regulator of cell growth, proliferation, and survival by interacting with various proteins. To date, few studies implicated mTOR in placenta. Human placenta in gestational diabetes mellitus (GDM) shows several alterations including villous immaturity, impaired placental function, and overgrowth. Hence, we aimed to investigate the expression of mTOR, phospho-mTOR (p-mTOR), and the 2 phosphorylated downstream targets of mTOR, ribosomal protein S6 kinase 1 (p-p70S6K), and eukaryotic initiation factor 4E-binding protein 1 (p-4EBP1) in normal term and gestational diabetic human placentas. Immunohistochemistry and Western blot were performed with antibodies against mTOR, p-mTOR, p-p70S6K, and p-4EBP1 (Thr37/46) in normal and diabetic placentas (n = 6 each) and quantified by ImageJ. All mTOR pathway components that we studied were immunolocalized in both normal and diabetic placenta groups. Syncytiotrophoblast and the vascular wall in villi displayed cytoplasmic mTOR and p-mTOR (S2448) immunoreactivities in all placenta samples. However, increased expression of p70S6K in syncytiotrophoblast and p-4EBP1 (Thr37/46) in villous stromal cells was observed in gestational diabetic placentas. Western blot analysis also confirmed the statistically significant increase in p-p70S6K (T389) expression in diabetic placentas. The altered expression of downstream components of mTOR signaling in gestational diabetic placentas suggests an involvement of mTOR activity in the placental pathology of GDM. However, whether increased nutrient transport via this pathway will stimulate fetal and placental overgrowth is still unknown. Although this is a descriptive study, further studies with a functional analysis to highlight the molecular mechanisms underlying this placental pathology are proposed.

Adenomyosis (ADS) is a common estrogen-dependent gynecological disease with unknown etiology. The RhoA/Rho-kinase (ROCK) signaling pathway is involved in various cellular functions, including migration, proliferation, and smooth muscle contraction. Here we examined the potential role of this pathway in junctional zone (JZ) contraction in women with and without ADS. We demonstrated that in the normal JZ, RhoA and ROCK-I messenger RNA (mRNA) and protein expression was significantly higher in the proliferative phase of the menstrual cycle than in the secretory phase. Expression of RhoA and ROCK-I in the JZ from women with ADS was significantly higher than in the control women and showed no significant differences across the menstrual cycle. Treatment of JZ smooth muscle cells (JZSMCs) with estrogen at 0, 1, 10, or 100 nmol/L for 24 hours resulted in increased expression of RhoA, ROCK-I, and myosin light-chain (MLC) phosphorylation (p-MLC) in a dose-dependent manner. In parallel to its effects on p-MLC, estrogen-mediated, dose-dependent contraction responses in JZSMCs. Estrogen-mediated contraction in the ADS group was significantly higher than in the controls and also showed no significant differences across the menstrual cycle. These effects were suppressed in the presence of ICI 182780 or Y27632, supporting an estrogen receptor-dependent and RhoA activation-dependent mechanism. Our results indicate that the level of RhoA and ROCK-I increases in patients with ADS and the cyclic change is lost. Estrogen may affect uterine JZ contraction of ADS by enhancing RhoA/ ROCK-I signaling.

This study evaluated the potential protective effect of the antioxidants, l-carnitine (LC) and N-acetyl-cysteine (NAC), in preventing meiotic oocyte damage induced by follicular fluid (FF) from infertile women with mild endometriosis (ME). We performed an experimental study. The FF samples were obtained from 22 infertile women undergoing stimulated cycles for intracytoplasmic sperm injection (11 with ME and 11 without endometriosis). Immature bovine oocytes were submitted to in vitro maturation (IVM) divided into 9 groups: no-FF (No-FF); with FF from control (CFF) or ME (EFF) groups; and with LC (C + LC and E + LC), NAC (C + NAC and E + NAC), or both antioxidants (C + 2Ao and E + 2Ao). After IVM, oocytes were immunostained for visualization of microtubules and chromatin by confocal microscopy. The percentage of meiotically normal metaphase II (MII) oocytes was significantly lower in the EFF group (51.35%) compared to No-FF (86.36%) and CFF (83.52%) groups. The E + NAC (62.22%), E + LC (80.61%), and E + 2Ao (61.40%) groups showed higher percentage of normal MII than EFF group. The E + LC group showed higher percentage of normal MII than E + NAC and E + 2Ao groups and a similar percentage to No-FF and CFF groups. Therefore, FF from infertile women with ME causes meiotic abnormalities in bovine oocytes, and, for the first time, we demonstrated that the use of NAC and LC prevents these damages. Our findings elucidate part of the pathogenic mechanisms involved in infertility associated with ME and open perspectives for further studies investigating whether the use of LC could improve the natural fertility and/or the results of in vitro fertilization of women with ME.

Suboptimal cellular conditions result in the accumulation of unfolded proteins in the endoplasmic reticulum (ER) and trigger ER stress. In this study, we investigated the effects of follicle stimulating hormone (FSH) on ER stress in granulosa cells (GCs) obtained from 3-week-old female C57BL6 mice 24 or 48 hours after intraperitoneal injection of 5 IU pregnant mare’s serum gonadotropin (PMSG), and in primary mouse GCs in culture treated with FSH (10-100 mIU/mL) for 24 or 48 hours. Moreover, mouse GCs in culture were treated with tunicamycin (Tm) or thapsigargin (Tp), which induce ER stress by inhibiting N-glycosylation of ER proteins and ER calcium adenosine triphosphatase, respectively, and their response to FSH was evaluated. We found that FSH attenuated ER stress in mouse GCs in vivo and in vitro; messenger RNA levels of ER stress-associated genes Xbp1s, Atf6, Chop, and Casp12 were decreased upon exposure to FSH/PMSG. Activating transcription factor 4 protein levels also demonstrated consistent decrease following FSH stimulation. Both Tm and Tp treatments inhibited FSH response, ER stress-induced cells did not show any change in estradiol levels in response to FSH, whereas in untreated GCs, estradiol production increased 3-fold after incubation with FSH for 60 hours. Furthermore, ER stress-induced cells failed to demonstrate aromatase (Cyp19a1) expression upon exposure to FSH. Importantly, under high-ER stress conditions FSH stimulation was unable to downregulate the expression of ER stress-associated genes. Our findings suggest that FSH decreases ER stress in GCs under physiologic conditions. However, under conditions that cause a significant increase in ER stress, FSH response is attenuated.

The effects of gestational diabetes mellitus (GDM) were determined on urinary excretion of putative components of insulin signaling. Random urine samples were collected from 375 gravidas at 6 to 14 weeks’ gestation, 22 to 32 weeks’ gestation, and ∼6 weeks’ postpartum. Gestational diabetes mellitus developed in 35 women who were matched with 59 normal gravidas. Urinary concentrations of myo-inositol (MI) and D-chiro-inositol (DCI) were measured by gas chromatography/mass spectrometry and normalized to creatinine levels. Compared to postpartum values, urinary excretion of MI and DCI was increased 2.9-fold and 2-fold, respectively, in early pregnancy, and 5.5-fold and 4.5-fold, respectively, in later gestation. Gravidas with GDM had significantly greater MI and DCI excretion than controls in the first trimester but not subsequently. The results suggest that gravidas destined to develop GDM have altered synthesis, metabolism, and/or renal excretion of MI and DCI in early pregnancy.

To evaluate the effect of atmospheric oxygen (O₂) concentration on embryonic development, a controlled and randomized study using the sibling oocytes was carried out. A total of 147 patients were studied. Embryos were cultured in O₂ concentration 20% versus 5% during the gamete, zygote, and first 3 days. The mean cell numbers of embryo (7.69 ± 1.91 vs 7.20 ± 1.82, P = .011) and rate of clinically useable embryos (81.62% vs 77.22%, P = .017) were significantly higher in 5% O₂ than in 20% O₂. There was no difference in the zygote developmental stage, day 2, day 4, and blastocyst stage. The quality of blastocyst (both inner cell mass and trophectoderm) showed no difference. Also, there was no increase in embryos fragmentation and uneven cells in 20% O₂ culture condition. In conclusion, 20% O₂ reduced the mean cell numbers of embryo and the number of clinically useable embryos on day 3. However, there was no subsequent negative impact on development of day 4 and blastocyst stage.

The objective was to investigate the expression of the WNT4 gene in ectopic endometrium and eutopic endometrium (EU) during endometriosis and the relationship of WNT4 expression with the menstrual cycle. Ectopic endometrium and EU tissues were collected from 30 women with pathologically confirmed endometriosis and 30 women without endometriosis. The WNT4 protein and messenger RNA (mRNA) expression levels were measured by fluorescence-based quantitative real-time polymerase chain reaction, immunohistochemistry, and Western blot methods. The expression of WNT4 was not significantly correlated with the menstrual cycle, and there were no significant differences when WNT4 expression in proliferative endometrium was compared with that in secretory endometrium within each group. There were no significant differences between the protein and mRNA expression of WNT4 in ectopic endometrium and in EU from participants with endometriosis. The WNT4 expression level in EU was significantly reduced compared with that in normal endometrium of the control group, even when analyzed by the menstrual cycle phase. WNT4 was also downregulated in ectopic lesions. This study provides further evidence supporting the theory of “EU determinism” in the pathogenesis of endometriosis.

Intrauterine adhesions (IUAs), which are characterized by endometrial fibrosis, increase the risk of secondary infertility and recurrent miscarriage. MicroRNA-29 (miR-29) is a potent inhibitor of TGF-β1/Smad signaling. In this study, we investigated the therapeutic potential of agomir-29b, an miR-29b mimic, in endometrial fibrosis induced by dual injury (uterine curettage and lipopolysaccharide treatment) in a rat model of IUA and explored the underlying mechanism. We found that injured rats developed endometrial fibrosis characterized by increased COL1A1 and α-smooth muscle actin expression and decreased E-cadherin expression, associated with a loss of miR-29b. Overexpression of miR-29b before injury prevented endometrial fibrosis including collagen accumulation and epithelial–mesenchymal transition. Delay of agomir-29b treatment until endometrial fibrosis was established on day 4 also halted the progression of disease. Further experiments indicated that miR-29b inhibited endometrial fibrosis via blockade of the Sp1-TGF-β1/Smad-CTGF pathway. In conclusion, agomir-29b may act as a novel and effective therapeutic agent against IUAs.

The association of intercellular adhesion molecule 1 (ICAM-1) genetic polymorphisms with uterine cervical carcinogenesis has seldom been reported. Therefore, the aim of this study was to investigate the association of single-nucleotide polymorphisms (SNPs) and haplotypes of ICAM-1 with cervical tumorigenesis in Taiwanese women. Four hundred forty four women, including 91 with cervical invasive cancer, 63 with precancerous lesions, and 290 normal controls, were recruited. The genotypic distribution of 4 SNPs of ICAM-1, rs5498 (A1548G), rs5491 (K56M), rs281432 (C8823G), and rs3093030 (C-286T) was determined using real-time polymerase chain reactions and genotyping. Compared to homozygous wild CC, heterozygous CG, homozygous mutant GG, or genotypes with CG/GG display increased risks or a tendency of precancerous lesions or invasive cancer with strong power in rs281432. The homozygotic mutant alleles TT in rs3093030 and homozygotic mutant alleles GG in rs5498 were associated with a higher risk of invasive cancer and precancerous lesions, respectively, but with lower power. The CG/TA/TG haplotypes of ICAM-1 SNPs rs3093030 and rs5498 exhibited a tendency to increase susceptibility to precancerous lesions and invasive cancer. In conclusion, Taiwanese women with ICAM-1 SNP rs281432 and haplotypes CG/TA/TG of rs3093030 and rs5498 are associated with uterine cervical carcinogenesis.