Follicular microenvironment: Oxidative stress and adiponectin correlated with steroids hormones in women undergoing in vitro fertilization.

Research has been focused on determining the follicular microenviroment produced by the theca and granulosa cells since the molecular characterisation of this body fluid could lead to the understanding of several fertility problems. Oxidative stress may be one of the factors involved in female infertility since it plays a key role in the modulation of oocyte maturation and finally pregnancy. An increase in oxidative stress is correlated with inflammation and intense research was developed to understand the interaction between inflammation and adiponectin, based on the fact that many adipokines are inflammation related proteins linked to reactive oxygen species production. The aim of this study is to investigate the correlation between total adiponectin levels and oxidative stress amount in the serum and follicular fluid (FF) of women who undergone in vitro fertilization. Moreover we verified the expression of adiponectin in granulosa and cumulus cells. To clarify the predictive value of steroid hormones in human assisted reproduction, twelve steroid hormones in FF and serum, were quantified in a single run liquid chromatography/mass spectrometry, by using a multiple reaction monitoring mode and we related the serum and follicular fluids adiponectin levels with the concentration of the investigated steroid hormones.

Management of perimenopause disorders: hormonal treatment.

Perimenopause represents a transition period of a woman's life during which physiological, affective, psychological, and social changes mark progression from a woman's fertile life to menopause, with wide sexual hormones fluctuations until the onset of hypergonadotropic hypogonadic amenorrhea. Contraception during menopause should not only avoid unwanted pregnancies, but also improve quality of life and prevent wide range of condition affecting this population. Hormonal contraceptives confer many noncontraceptive benefits for women approaching menopause: treatment of abnormal uterine bleeding, relief from vasomotor symptoms, endometrial protection in women using estrogen therapy, musculoskeletal protection, and mood disorders protection. The main point remains selecting the most adequate contraceptive option for each woman, considering her risk factor, comorbidities, and keeping in mind the possibility of continuing contraception until reaching menopause and even further, creating a bridge between perimenopause and menopause hormonal therapy. Correct perimenopause management should rely on individualized medical therapy and multidisciplinary approach considering lifestyle and food habits as part of general good health of a woman.

Ulipristal acetate before in vitro fertilization: efficacy in infertile women with submucous fibroids.

BACKGROUND: The presence of submucous fibroids strongly impacts on IVF results, therefore, these patients should be considered for surgical or medical treatment. The aim of this study was to assess the role of Ulipristal acetate (UPA), a selective progesterone receptor modulator, in restoring uterine cavity deformation due to submucous fibroids, in infertile patients attempting an IVF treatment. The secondary study outcome was to evaluate the impact of preconception UPA treatment on rate of biochemical pregnancy, ongoing pregnancy, and live birth compared to a control group without fibroids. METHODS: Infertile patients with submucosal fibroid (Type 1 and Type 2 according to FIGO classification) were enrolled in the study as fibroids group and received 1 to 3 treatment cycles of UPA, according to their response, as reflected by fibroid volume reduction and restoration of normal uterine cavity. Patients in control group were randomly selected from a general IVF cohort by a ratio of 2:1 with fibroids group, matched by age, BMI, type and cause of infertility and antral follicle count. The impact of UPA on fibroids volume reduction was evaluated. IVF outcome was compared between groups. RESULTS: Twenty-six patients underwent UPA treatment revealed a mean volume reduction of their fibroids of 41%. A total of 15 (57.6%) biochemical pregnancy were obtained, resulting in 13 (50%) ongoing pregnancy and 9 (34.6%) healthy babies were already delivered. Similar results were obtained in control group. CONCLUSION: Restoration of normal uterine cavity by UPA treatment prior to IVF treatment avoids surgery and establishes a pregnancy rate comparable to a control group without fibroids.

Evaluation of different antiandrogenic progestins on clinical and biochemical variables in polycystic ovary syndrome.

Objectives: The aim of the study was to update the results of a previous study published 10 years ago and compare the effect on hyperandrogenism of a newer progestin, dienogest (DNG), in a combined oral contraceptive (COC) formulation with ethinylestradiol (EE), with that of COCs containing the same dose of EE in combination with drospirenone (DRSP) and chlormadinone acetate (CMA).Methods: Sixty women with polycystic ovary syndrome (PCOS) aged between 16 and 35 and requiring antiandrogenic contraceptive treatment were randomised to one of three treatment groups: EE 30 µg/DRSP 3 mg, EE 30 µg/CMA 2 mg, EE 30 µg/DNG 2 mg. We evaluated the effects of the three COCs on sex hormone-binding globulin (SHBG) and biochemical markers of hyperandrogenism.Results: After 3 months of treatment, serum androgen concentrations were significantly improved in all treatment groups. Serum concentrations of SHBG were significantly increased with all COC treatments (p < 0.0001). Interestingly, DRSP had a greater effect (+218%; p < 0.0001) on serum SHBG concentrations compared with DNG and CMA (p < 0.04 and p < 0.002, respectively). Serum concentrations of total testosterone significantly decreased in all groups (p < 0.0001). DRSP had a significantly greater effect on total testosterone concentrations compared with DNG (p = 0.002) and CMA (p < 0.0001).Conclusion: Our study showed that DNG exerted an important stimulatory effect on SHBG concentrations, which was less than that of DRSP but greater than that of CMA. Similar results were also obtained for dehydroepiandrosterone sulphate and total testosterone.

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.

Alterations in thyroid function among the different polycystic ovary syndrome phenotypes.

The study evaluates the prevalence of subclinical thyroid dysfunction in infertile PCOS patients, according to the different PCOS phenotypes and to examine whether insulin sensitizers in insulin resistant (IR) PCOS patients may improve thyroid function. The study population consisted of all PCOS patients, attending the infertility and IVF unit of Department of Pediatrics, Obstetrics and Reproductive Medicine of University of Siena, Italy, and compared them to regularly cycling, healthy, infertile controls. Upon admission, blood was drawn from all patients during the early follicular phase, for complete hormonal and metabolic profiles. In IR-PCOS patients treated with insulin sensitizers, blood was drawn again after 6 months. PCOS patients had a significantly higher prevalence of subclinical thyroid dysfunction compared to infertile controls. While no significant association was detected between TSH value and the presence of hyperandrogenism, overweight and obese PCOS patients, as well as IR PCOS patients showed significantly higher prevalence of subclinical thyroid dysfunction. Moreover, among IR PCOS patients, 6 months treatment with insulin sensitizers significantly reduces TSH levels. Infertile PCOS patients have a high prevalence of subclinical thyroid dysfunction, which may be successfully treated in IR PCOS patients by insulin sensitizers.

Positive Effect of a New Combination of Antioxidants and Natural Hormone Stimulants for the Treatment of Oligoasthenoteratozoospermia.

Oligoasthenoteratozoospermia (OAT) accounts for about 90% of male infertility; in many cases this disorder may be associated with oxidative stress, a condition that decreases the success of fertilization. Therefore, the empirical treatment of male infertility is often based on the use of antioxidants. The aim of the present study was to assess the effectiveness of three months' administration of a new nutraceutical preparation on hormone profile, sperm parameters and fertilization capability in men undergoing in vitro fertilization (IVF). A total of 36 OAT patients were daily treated for 3 months with a dose of a formulation containing: Inositol, L-Carnitine, Vitamins C, D, E, Coenzyme Q10 and Selenium. Selected parameters were analysed before (T0) and after (T1) treatment, and IVF outcomes were evaluated. We observed an improvement of sperm concentration, motility, morphology and vitality; blood level of testosterone also showed an increase. A significant increase of fertilization rate was detected in 14 couples, whose male partner were treated with the nutraceutical preparation. The present results indicate that a formulation containing antioxidant and energy supply substances was effective in the treatment of sperm alterations and led to significant recovery of fertilizing capacity.

Human Polymorphonuclear Cells Support Zika Virus to Cross Endothelial Monolayer and Access Bloodstream.

The rapid spread of new outbreaks of human infection caused by Zika virus (ZIKV) has raised many global concerns since 2016. Despite the increasing knowledge of this virus, data on the pathogenesis of ZIKV are still missing. In particular, it is still unknown how the virus crosses the endothelial monolayer and gets access to the bloodstream. In the present work, we used human umbilical vein endothelial cells (HUVECs) as a model to study ZIKV infection in vitro. We demonstrated that HUVECs are an optimal reservoir for viral replication, as they were able to sustain ZIKV infection up to two weeks, without showing a cytopathic effect. In order to evaluate the integrity of endothelial monolayer, immunofluorescence was performed on mock-infected or ZIKV-infected cells ± peripheral blood mononuclear cells (PBMCs) or polymorphonuclear cells (PMN), 48 h p.i., by using an anti-VE-Cadherin antibody, a major adherence protein that maintains the integrity of intercellular junctions. In addition to infection, we noted that the presence of some components of the immune system, such as PMNs, played an important role in altering the endothelial monolayer in cell junctions, suggesting that presence at the site of infection probably promotes the spread of ZIKV in vivo in the bloodstream.

SARS-CoV-2 Infection of Human Ovarian Cells: A Potential Negative Impact on Female Fertility.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) may affect female reproductive health. Here, we investigated the potential of SARS-CoV-2 to infect the follicular microenvironment, in particular granulosa (GCs) and cumulus cells (CCs), thus providing evidence for a productive infection. GCs and CCs were recovered from women (n = 25) who underwent in vitro fertilization at the Assisted Reproductive Unit, Siena University Hospital. Follicular ovarian cells were co-cultured with SARS-CoV-2 and then analyzed by qPCR, immunofluorescence (IF), western blot (WB) and transmission electron microscopy (TEM). In addition, cell culture supernatant was used to infect VERO6 cells. We demonstrated the expression of cell host factors ACE2, TRPMSS2, BSG and CTSL, which are pivotal for the virus life cycle. Cultured GCs and CCs incubated with SARS-CoV-2 revealed productive SARS-CoV-2 infection at 24 h, 48 h and 72 h post-adsorption. Indeed, SARS-CoV-2 RNA, spike and nucleocapsid proteins were detected in GCs and CCs, and their cell culture supernatant successfully infected the standard VERO E6 cells. Finally, TEM showed full-size virions attached to the membrane and located inside the cytoplasm. This in vitro study reveals the susceptibility of human ovarian cells to SARS-CoV-2 infection, suggesting a potential detrimental effect of COVID-19 infection on female human fertility.

PCOS Physiopathology and Vitamin D Deficiency: Biological Insights and Perspectives for Treatment.

Recent literature has stressed the importance of vitamin D (VD) in polycystic ovary syndrome (PCOS). Women with PCOS are deficient in VD, particularly those with a higher weight. Hypovitaminosis is a risk factor for glucose intolerance, and reduced levels of VD is associated with insulin resistance and increased diabetes risk. Since women with PCOS and hirsutism seem to have lower levels of VD than women with PCOS without hirsutism, a correlation between VD deficiency and hyperandrogenism may be suggested. Interestingly, VD is crucial for many human physiological functions, including to counteract inflammation and oxidative stress. Some studies evaluated effects of VD supplementation on glucose homeostasis variables, hormonal status, lipid concentrations, and biomarkers of inflammation and oxidative stress among VD-deficient women. Moreover, VD has been shown to play a role in egg quality and fertility. This review aims to show the relationship between VD and the endocrine and metabolic profile of PCOS patients, as well as its implications for their fertility. The supplement of VD to the common therapy can lead to an improvement of the insulin resistance and lipid metabolism, a reduction of circulating androgens, as well as a better response to the induction of ovulation in PCOS women.

Coronavirus Disease 2019 (SARS-CoV-2) and polycystic ovarian disease: Is there a higher risk for these women?

The new severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been associated with acute respiratory distress syndrome and infected patients have a relatively high risk of death. Emerging risk factors for poor outcome in this disease include age, male gender, cardiovascular co-morbidities including hypertension, prior cardiovascular disease, diabetes and more recently obesity. To date there are no data relating to SARS-CoV-2 in PCOS women. The present Clinical Opinion represents a summary of the epidemiological evidence and possible pathophysiological mechanisms regarding PCOS and COVID-19. PCOS women could be more susceptible to infections compared to non-PCOS women. Insulin resistance and the associated hyperinsulinaemia are drivers for enhanced steroidogenesis in women with PCOS. Weight-gain and obesity, through their worsening effects on insulin resistance, thereby drive enhanced steroidogenesis and hyperandrogenism. All these features represent key points to provide an explanation for the possible association between PCOS and SARS-CoV-2. Indeed, androgens may drive clinical results in COVID-19, through the expression of TMPRSS2, a cellular co-receptor necessary for SARS-CoV-2 infection and through androgen-mediated immune modulation. In women with PCOS the endocrine-immune axis leads to immune dysfunction with a state of chronic inflammation, and hyperandrogenism and IR with compensatory hyperglycaemia could play a determining role in the pathophysiogenesis of the infection. However, it is possible that only specific PCOS phenotypes may be more susceptible. In addition, vitamin D deficiency and gut dysbiosis are another important factor potentially involved in the increased risk of developing severe forms of COVID-19 in PCOS women. Further scientific investigations are needed with the aim of understanding which women are most at risk of becoming infected or developing complications, what are the causal mechanisms on which it is possible to intervene with prophylactic and therapeutic measures and what the long-term consequences will be on the health of these patients.

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.

Bitter Taste Receptors Expression in Human Granulosa and Cumulus Cells: New Perspectives in Female Fertility.

Bitter taste receptors (TAS2RS) expression is not restricted to the oral cavity and the presence of these receptors in the male reproductive system and sperm provides insights into their possible role in human reproduction. To elucidate the potential role of TAS2Rs in the female reproductive system, we investigated the expression and localization of bitter taste receptors and the components of signal transduction cascade involved in the pathway of taste receptors in somatic follicular cells obtained from women undergoing assisted reproductive techniques. We found that TAS2R genes are expressed in both cumulus (CCs) and granulosa (GCs) cells, with TAS2R14 being the most highly expressed bitter receptor subtype. Interestingly, a slight increase in the expression of TAS2R14 and TAS2R43 was shown in both GCs and CCs in young women (p < 0.05), while a negative correlation may be established between the number of oocytes collected at the pickup and the expression of TAS2R43. Regarding α-gustducin and α-transducin, two Gα subunits expressed in the taste buds on the tongue, we provide evidence for their expression in CCs and GCs, with α-gustducin showing two additional isoforms in GCs. Finally, we shed light on the possible downstream transduction pathway initiated by taste receptor activation in the female reproductive system. Our study, showing for the first time the expression of taste receptors in the somatic ovarian follicle cells, significantly extends the current knowledge of the biological role of TAS2Rs for human female fertility.

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.

Organoids of Human Endometrium: A Powerful In Vitro Model for the Endometrium-Embryo Cross-Talk at the Implantation Site.

Embryo implantation has been defined as the "black box" of human reproduction. Most of the knowledge on mechanisms underlining this process derives from animal models, but they cannot always be translated to humans. Therefore, the development of an in vitro/ex vivo model recapitulating as closely and precisely as possible the fundamental functional features of the human endometrial tissue is very much desirable. Here, we have validated endometrial organoids as a suitable 3D-model to studying epithelial endometrial interface for embryo implantation. Transmission and scanning electron microscopy analyses showed that organoids preserve the glandular organization and cell ultrastructural characteristics. They also retain the responsiveness to hormonal treatment specific to the corresponding phase of the menstrual cycle, mimicking the in vivo glandular-like aspect and functions. Noteworthy, organoids mirroring the early secretive phase show the development of pinopodes, large cytoplasmic apical protrusions of the epithelial cells, traditionally considered as reliable key features of the implantation window. Moreover, organoids express glycodelin A (GdA), a cycle-dependent marker of the endometrial receptivity, with its quantitative and qualitative features accounting well for the profile detected in the endometrium in vivo. Accordingly, organoids deriving from the eutopic endometrium of women with endometriosis show a GdA glycosylation pattern significantly different from healthy organoids, confirming our prior data on endometrial tissues. The present results strongly support the idea that organoids may closely recapitulate the molecular and functional characteristics of their cells/tissue of origin.

Sperm aneuploidies after human recombinant follicle stimulating hormone therapy in infertile males.

Errors in sperm chromosome segregation are frequently observed in infertile males. It would therefore be useful to develop methods for reducing the rate of aneuploidy in spermatozoa. Thirty-one males were selected with an elevated frequency of total sperm aneuploidy of sperm chromosomes 18, X and Y by fluorescence in-situ hybridization (FISH): 22 were treated with 150 IU of recombinant FSH on alternate days for 3 months and the other nine (controls) did not receive any hormonal treatment. Before therapy, FISH analysis demonstrated an increased frequency of diploidy (0.663 +/- 0.09%), disomy (0.412 +/- 0.03%) and total aneuploidy (1.30 +/- 0.12%) in the 22 males. Sperm analyses revealed reduced progressive motility (26.73 +/- 2.3%) and a reduced percentage of spermatozoa with normal morphology (23.86 +/- 5.3%). After 90 days of therapy, a significant reduction in aneuploidies (mean total aneuploidy: 0.86% +/- 0.11; P = 0.005) was obtained, as well as an improvement in functional and structural sperm characteristics. In untreated patients, no significant change in semen parameters and frequency of total aneuploidy was observed between baseline (1.054 +/- 0.06%) and 90 days later (1.080 +/- 0.05%). It is therefore suggested that deranged meiotic segregation in spermatozoa could be reduced by FSH treatment.

Sperm quality improvement after natural anti-oxidant treatment of asthenoteratospermic men with leukocytospermia.

AIM: To study the immune-modulating and anti-oxidant effects of beta-glucan, papaya, lactoferrin, and vitamins C and E on sperm characteristics of patients with asthenoteratozoospermia associated with leucocytosis. METHODS: Fifty-one patients referred to our Sterility Center for semen analysis were selected. Sperm parameters were assessed before and after patient's treatment with beta-glucan, lactoferrin, papaya, and vitamins C and E. DNA damage was assessed by the acridine orange test and sperm structural characteristics were evaluated by transmission electron microscopy. RESULTS: After 90 days of treatment, an increase in the percentage of morphologically normal sperm (17.0 +/- 5.2 vs. 29.8 +/- 6.5) and total progressive motility (19.0 +/- 7.8 vs. 34.8 +/- 6.8) were detected. Structural sperm characteristics as well as chromatin integrity were also improved after treatment. In terms of leukocyte concentration in seminal fluid, a significant reduction was recorded (2.2 +/- 0.9 vs. 0.9 +/- 0.2). CONCLUSION: The treatment of an inflammatory process by the synergic action of immune modulators and anti-oxidants could protect sperm during maturation and migration, leading to improved sperm function.

[The role of vitamin D in assisted reproduction techniques].

Vitamin D or calcitriol, which was first identified back in 1920, has a key role in bone metabolism and mineralization, and in calcium and magnesium homeostasis. A nuclear receptor (VDR) mediates vitamin D actions in a lot of organs like bowel, bone, kidney, breast, gonads, pancreas, brain, cardiovascular and immune systems. In all these tissues the 1,25(OH)2D play a fundamental role in reduction of chronic pathologies, in modulation of cellular growth, neuromuscular, immune and inflammation actions. Vitamin D is important in reproduction physiology and in development gynecological and obstetric disorders like polycystic ovarian syndrome, endometriosis, myomatosis, gestational diabetes and preeclampsia. Over the last few years, vitamin D has been suggested to have a role in assisted reproduction techniques.

Mental health, pain symptoms and systemic comorbidities in women with endometriosis: a cross-sectional study.

INTRODUCTION: Endometriosis is a gynecological disease with a severe impact on quality of life. The aim of this study is to assess mental health status in a group of women with endometriosis, investigating their clinical history, pain symptoms and systemic comorbidities. METHODS: An observational cross-sectional study was performed on a group of Italian Caucasian women with endometriosis (n = 134). All patients filled the 'Patient Health Questionnaire' (PHQ), a self-administered screening tool for mental health disorders. The characteristics of endometriosis, pain symptoms and their severity, the presence and types of comorbid systemic disorders were added into the same survey. RESULTS: According to PHQ algorithms, 59% of patients were affected by at least one psychiatric disorder, with a significant correlation with pain symptoms (p = 0.0026). Patients with severe pain showed a higher incidence of multiple psychiatric disorders (p = 0.026) and somatoform disorder than those with mild pain (p = 0.0009). There was no correlation between the presence of psychiatric disorders and age, BMI, parity, infertility, need for surgery, number of intervention, localization of endometriotic lesions and systemic comorbidities. DISCUSSION: Women with endometriosis showed a high frequency of PHQ results positive for psychiatric disorders, with a significant association with pain severity.

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.