Emerging role of embryo secretome in the paracrine communication at the implantation site: a proof of concept.

OBJECTIVE: To assess the role of embryo secretome in modifying the molecular profile of glycodelin A (GdA) in endometrial organoids (ORG) mimicking the implantation window. To verify whether the use of embryo-conditioned culture medium at the time of the embryo transfer may increase in vitro fertilization outcome. DESIGN: Molecular study with human endometrial ORG and embryo-conditioned culture medium. Retrospective study using prospectively recorded data. SETTING: University hospital. PATIENT(S): For isolation and culture of endometrial glandular ORG, endometrial biopsy specimens from five white women of proven fertility undergoing laparoscopy for tubal sterilization. A total of 75 women undergoing intracytoplasmic sperm injection for tubal and/or male infertility factor. INTERVENTIONS(S): In vitro fertilization. MAIN OUTCOME MEASURE(S): Pinopodes presence in human endometrial ORG. Glycodelin A expression profile by means of two-dimensional electrophoresis. In vitro fertilization outcome. RESULT(S): This in vitro study demonstrated that the treatment of endometrial ORG with the secretome of medium conditioned by the growing embryo increased the GdA relative abundance and induced a different glycoform pattern. Biochemical and clinical pregnancy rate significantly increased when the spent medium was loaded during the transfer (17.5% vs. 36.6% and 16.5% vs. 35.1%, respectively). CONCLUSION(S): This study demonstrated that the secretome of implanting embryos is able to induce the expression as well as to determine the relative abundance and the glycosilation profile of endometrial GdA, a protein having a key role in the embryo-endometrial cross talk. Moreover, a significant increase in pregnancy rate was observed when the embryo transfer was performed by using the culture medium conditioned by the growing embryo.

Characterization of the Age-Dependent Changes in Antioxidant Defenses and Protein's Sulfhydryl/Carbonyl Stress in Human Follicular Fluid.

The oxidative stress, characterized by the imbalance between pro-oxidants and antioxidants molecules, seems to be involved in the pathogenesis of female subfertility. In particular, the presence of different markers of oxidative stress has been reported in human follicular fluid (FF) surrounding oocytes. Based on its distinctive composition and on the close proximity to the oocyte, FF creates a unique microenvironment having a direct impact on oocyte quality, implantation, and early embryo development. An imbalance in reactive oxygen species (ROS) production in ovarian follicular fluid may have a negative effect on these processes and, as a consequence, on female fertility. Therefore, the aim of this study was to evaluate the redox state of the FF through various methodological approaches. By means of 2D-electrophoresis we demonstrated that the main structural changes occurring in the proteins of the follicular fluid of normovulatory women were correlated to the age of the patients and to the antioxidant defenses present in the FF. Measurement of these parameters could have clinical relevance, since the assessment of the oxidative stress rate may be helpful in evaluating in vitro fertilization potential.

Respiratory Mitochondrial Efficiency and DNA Oxidation in Human Sperm after In Vitro Myo-Inositol Treatment.

Semen samples are known to contain abnormal amounts of reactive oxygen species (ROS) and oxygen free radicals; therefore, the identification of antioxidant molecules able to counteract the oxidative damage caused by ROS is foresight. Indeed, improving semen quality in terms of motility and reduction in DNA damage, can significantly improve the fertilization potential of sperm in vitro. To this regard, myo-inositol, based on its antioxidant properties, has been reported to be effective in improving sperm quality and motility in oligoasthenozoospermic patients undergoing assisted reproduction techniques when used as a dietary supplementation. Moreover, in vitro treatment demonstrated a direct relationship between myo-inositol, mitochondrial membrane potential and sperm motility. This experimental study aimed to evaluate the effects of myo-inositol (Andrositol-lab) in vitro treatment on sperm motility, capacitation, mitochondrial oxidative phosphorylation and DNA damage. Our results demonstrate that myo-inositol induces a significant increase in sperm motility and in oxygen consumption, the main index of oxidative phosphorylation efficiency and ATP production, both in basal and in in vitro capacitated samples. Moreover, we provide evidence for a significant protective role of myo-inositol against oxidative damage to DNA, thus supporting the in vitro use of myo-inositol in assisted reproductive techniques. Even if further studies are needed to clarify the mechanisms underlying the antioxidant properties of myo-inositol, the present findings significantly extend our knowledge on human male fertility and pave the way to the definition of evidence-based guidelines, aiming to improve the in vitro procedure currently used in ART laboratory for sperm selection.

Increased expression of neurogenic factors in uterine fibroids.

STUDY QUESTION: Are selective markers for the neuronal differentiation such as microtubule-associated protein 2 (MAP-2) and synaptophysin (SYP) as well as the nerve growth factor (NGF) expressed by fibroids, myometrium and eutopic endometrium? SUMMARY ANSWER: Neuronal markers NGF, MAP-2 and SYP are highly expressed in fibroids compared with matched myometrium, and this neurogenic pathway is upregulated by tumor necrosis factor (TNF) alpha in cultured smooth muscle cells (SMCs). WHAT IS KNOWN ALREADY: Uterine fibroids or leiomyomas are the most common benign tumors, accounting for approximately one-third of hysterectomies. The present trend is to improve the medical treatment avoiding surgery, also for fertility sparing; hence, the pathogenic mechanisms are investigated, aiming to develop new therapeutic strategy. STUDY DESIGN, SIZE, DURATION: This laboratory-based case-control study is focused on fibroids and myometrial specimens obtained between 2015 and 2017 from 15 women of reproductive age at the proliferative phase of the menstrual cycle. Leiomyomas, matched myometrium and endometrium from each woman were analyzed. Control endometrium was obtained from women undergoing surgery for ovarian cyst (n = 15). PARTICIPANTS/MATERIALS, SETTING, METHODS: qRT-PCR, western blotting and immunostaining were applied to evaluate the expression of neurogenic markers; the effects of TNF on NGF, MAP-2 and SYP expression in cultured SMCs from leiomyomas and matched myometrium were analyzed. MAIN RESULTS AND THE ROLE OF CHANCE: qRT-PCR analyses using tissues from clinical patients showed that the levels of NGF, MAP-2 and SYP mRNA were significantly higher in uterine leiomyomas compared with their matched myometrium (P < 0.05), whereas only NGF was significantly increased in eutopic endometrium compared with healthy endometrium. In primary SMCs, isolated from fibroids or from the adjacent myometrium, NGF, MAP-2 and SYP mRNA expression were significantly increased by TNF treatment (P < 0.05). Finally, human endometrial stromal cells prepared from the endometrium of patients affected by uterine fibroids display higher TNF expression (P < 0.001). LIMITATIONS, REASONS FOR CAUTION: qRT-PCR analysis and immunofluorescence validation are robust methods demonstrating a clear upregulation of neurogenic factors in leiomyomas, even though additional studies are needed to establish a correlation between increased neuronal gene expression and degree of pain, as well as the involvement of inflammation mediators in the development of the neurogenic unhinge. Therefore, more in vivo studies are needed to confirm the results achieved from primary cultured SMCs. WIDER IMPLICATIONS OF THE FINDINGS: The increased expression of neurogenic factors in uterine fibroids and endometrium may contribute to explain the painful stimuli. Accordingly, these neurogenic pathways may represent potential therapeutic avenues to treat the fibroid-related disorders. STUDY FUNDING/COMPETING INTEREST(S): This study was supported by research grants from the University of Siena. The authors declare no conflict of interest. TRIAL REGISTRATION NUMBER: N/A.

Matrix metalloproteinases and their inhibitors in human cumulus and granulosa cells as biomarkers for oocyte quality estimation.

OBJECTIVE: To study the molecular profile of metalloproteinases and their tissue inhibitors in granulosa and cumulus cells in a subset of fertile and infertile women. DESIGN: Molecular study with granulosa and cumulus cells. SETTING: University hospital. PATIENT(S): Forty-four women undergoing assisted reproductive techniques for female infertility factor, with partners having a normal spermiogram and 15 normally fertile women with male partner affected by severe oligoasthenoteratozoospermia or nonobstructive azoospermia. INTERVENTION(S): In vitro fertilization. MAIN OUTCOME MEASUREMENT(S): We investigated gene expression level of metalloproteinases (MMP2, MMP9, MMP11) and their tissue inhibitors (TIMP1, TIMP2) by means of quantitative reverse-transcription polymerase chain reaction, protein quantification by means of Western blot, and localization by means of immunofluorescence. RESULT(S): We firstly validated HPRT1 as the most reliable housekeeping gene enabling correct gene expression analysis in both granulosa and cumulus cells. Gene expression, Western blot, and immunofluorescence analysis of MMP2, MMP9, and MMP11 and their tissue inhibitors TIMP1 and TIMP2 demonstrated that these enzymes are finely tuned in these cells. MMP9 is specifically expressed only in granulosa, whereas MMP2 is more expressed in cumulus and granulosa cells in cases of reduced ovarian response and decreased fertilization rate. CONCLUSION(S): This study sheds light on MMP and TIMP expression in granulosa and cumulus cells, and it may help in understanding the fine regulation of oocyte maturation inside the follicle. Although further studies are needed to fully understand the molecular mechanisms involved in these processes, our findings may be useful in the identification of biomarkers of oocyte maturation, competence acquiring, and fertilization.

Dysregulation of GdA Expression in Endometrium of Women With Endometriosis: Implication for Endometrial Receptivity.

Glycodelin-A (GdA) has been proposed to represent a potential biomarker of endometrial function, but little is known about its expression during the different phases of the menstrual cycle and under pathological conditions. In the light of its potential importance also in embryo implantation, we aimed to evaluate the expression profile of GdA as well as the presence of different glycosylated glycoforms and the immunolocalization in endometrial tissue from women with endometriosis and in women with proven fertility, at different times during the menstrual cycle. Our results showed that GdA is synthesized by endometrial epithelial and stromal cells, both in healthy endometrium and eutopic endometrium from women with endometriosis, with a profile including several glycosylated glycoforms, differentially expressed in each phase of the menstrual cycle. During the secretory phase, a significant increase in GdA protein expression, with a different glycoforms profile, was observed in endometriotic eutopic endometrium. Protein localization in eutopic endometrial tissue resulted significantly different in comparison with endometrium from women with proven fertility. This study indicate that GdA is a complex glycoprotein including up to 6 different glycoforms specifically expressed during the different phase of the menstrual cycle; in pathologic conditions such as endometriosis, the expression profile is altered possibly related to the impaired endometrial receptivity.

Suppression of galactocerebrosidase premature termination codon and rescue of galactocerebrosidase activity in twitcher cells.

Krabbe's disease (KD) is a degenerative lysosomal storage disease resulting from deficiency of ß-galactocerebrosidase activity. Over 100 mutations are known to cause the disease, and these usually occur in compound heterozygote patterns. In affected patients, nonsense mutations leading to a nonfunctional enzyme are often found associated with other mutations. The twitcher mouse is a naturally occurring model of KD, containing in ß-galactocerebrosidase a premature stop codon, W339X. Recent studies have shown that selected compounds may induce the ribosomal bypass of premature stop codons without affecting the normal termination codons. The rescue of ß-galactocerebrosidase activity induced by treatment with premature termination codon (PTC) 124, a well-characterized compound known to induce ribosomal read-through, was investigated on oligodendrocytes prepared from twitcher mice and on human fibroblasts from patients bearing nonsense mutations. The effectiveness of the nonsense-mediated mRNA decay (NMD) inhibitor 1 (NMDI1), a newly identified inhibitor of NMD, was also tested. Incubation of these cell lines with PTC124 and NMDI1 increased the levels of mRNA and rescued galactocerebrosidase enzymatic activity in a dose-dependent manner. The low but sustained expression of ß-galactocerebrosidase in oligodendrocytes was sufficient to improve the morphology of the differentiated cells. Our in vitro approach provides the basis for further investigation of ribosomal read-through as an alternative therapeutic strategy to ameliorate the quality of life in selected KD patients. © 2016 Wiley Periodicals, Inc.

Antioxidants reduce oxidative stress in follicular fluid of aged women undergoing IVF.

BACKGROUND: The status characterized by the imbalance between pro-oxidants and antioxidants molecules, defined as oxidative stress, has been suggested to be involved in the pathogenesis of subfertility in females. This study aims to evaluate the impact of a complete micronutrients supplementation on oxidative stress levels in follicular microenvironment as well as on in vitro fertilization (IVF) outcome. METHODS: This preliminary study was conducted between January 2014 and July 2015 at the Siena University Hospital Infertility Clinic. Serum and follicular fluid were collected from infertile women aged > 39 years who underwent two in vitro fertilization cycles: in the first cycle they were treated with GnRH-antagonist protocol and gonadotropins for controlled ovarian hyperstimulation, whereas in the second cycle ovarian stimulation protocol was associated to micronutrients supplementation, starting three months earlier. Protein oxidation levels and total antioxidant capacity in serum and in follicular fluid were evaluated in IVF cycles with or without micronutrients supplementation. Differences in IVF outcome parameters were statistically evaluated. RESULTS: Two-dimensional electrophoresis analyses demonstrated that when patients assumed micronutrients before IVF cycles, follicular fluid and serum proteins were protected from oxidative damage. Comparable results were obtained when total antioxidant capacity was measured. Moreover, the mean number of good quality oocytes retrieved when patients received micronutrients supplementation was significantly increased. CONCLUSION: The additional treatment with micronutrients, starting three months before IVF cycles, protects the follicular microenvironment from oxidative stress, thus increasing the number of good quality oocytes recovered at the pick up.