Preservation of fertility in female patients with hematologic diseases.

Recent developments of assisted reproduction techniques turned possible to avoid the infertility consequences of oncologic treatments, but fertility preservation (FP) has been somewhat neglected in women with hematologic diseases undergoing gonadotoxic treatments. For these specific cases, the current options for FP include the cryopreservation of embryos, mature oocytes and ovarian tissue, and oocyte in-vitro maturation. We intend to make patients and clinicians aware of this important and relevant issue, and provide hematologists, assisted reproduction physicians and patients, with updated tools to guide decisions for FP. The physicians of the units responsible for female FP should always be available to decide on the best-individualized FP option in strict collaboration with hematologists. With a wide range of options for FP tailored to each case, a greater level of training and information is needed among clinicians, so that patients proposed to gonadotoxic treatments can be previously advised for FP techniques in hematological conditions. ABBREVIATED ABSTRACT: Recent developments of assisted reproduction techniques turned possible to preserve the fertility of women with hematologic diseases undergoing gonadotoxic treatments. Current options for fertility preservation in women with hematologic diseases are presented. It is imperative to offer fertility preservation to all women before starting any gonadotoxic treatment and in some cases after treatment. Fertility preservation methods enable to later achieve the desired pregnancy.

Spatial Distribution of SARS-CoV-2 Receptors and Proteases in Testicular Cells.

Coronavirus disease (COVID-19) is caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). SARS-CoV-2 RNA has been found in the human testis on occasion, but subgenomic SARS-CoV-2 and infectious SARS-CoV-2 virions have not been found. There is no direct evidence of SARS-CoV-2 infection of testicular cells. To better understand this, it is necessary to determine whether SARS-CoV-2 receptors and proteases are present in testicular cells. To overcome this limitation, we focused on elucidating with immunohistochemistry the spatial distribution of the SARS-CoV-2 receptors angiotensin-converting enzyme 2 (ACE2) and cluster of differentiation 147 (CD147), as well as their viral spike protein priming proteases, transmembrane protease serine 2 (TMPRSS2) and cathepsin L (CTSL), required for viral fusion with host cells. At the protein level, human testicular tissue expressed both receptors and proteases studied. Both ACE2 and TMPRSS2 were found in interstitial cells (endothelium, Leydig, and myoid peritubular cells) and in the seminiferous epithelium (Sertoli cells, spermatogonia, spermatocytes, and spermatids). The presence of CD147 was observed in all cell types except endothelium and peritubular cells, while CTSL was exclusively observed in Leydig, peritubular, and Sertoli cells. These findings show that the ACE2 receptor and its protease TMPRSS2 are coexpressed in all testicular cells, as well as the CD147 receptor and its protease CTSL in Leydig and Sertoli cells, indicating that testicular SARS-CoV-2 infection cannot be ruled out without further investigation.

All that glitters is not gold: a stereological study of human donor oocytes.

Here we report a quantitative analysis of human metaphase II (MII) oocytes from a 22-year-old oocyte donor, retrieved after ovarian-controlled hyperstimulation. Five surplus donor oocytes were processed for transmission electron microscopy (TEM), and a stereological analysis was used to quantify the distribution of organelles, using the point-counting technique with an adequate stereological grid. Comparisons between means of the relative volumes (Vv) occupied by organelles in the three oocyte regions, cortex (C), subcortex (SC) and inner cytoplasm (IC), followed the Kruskal-Wallis test and Mann-Whitney U-test with Bonferroni correction. Life cell imaging and TEM analysis confirmed donor oocyte nuclear maturity. Results showed that the most abundant organelles were smooth endoplasmic reticulum (SER) elements (26.8%) and mitochondria (5.49%). Significant differences between oocyte regions were found for lysosomes (P = 0.003), cortical vesicles (P = 0.002) and large SER vesicles (P = 0.009). These results were quantitatively compared with previous results using prophase I (GV) and metaphase I (MI) immature oocytes. In donor MII oocytes there was a normal presence of cortical vesicles, SER tubules, SER small, medium and large vesicles, lysosomes and mitochondria. However, donor MII oocytes displayed signs of cytoplasmic immaturity, namely the presence of dictyosomes, present in GV oocytes and rare in MI oocytes, of SER very large vesicles, characteristic of GV oocytes, and the rarity of SER tubular aggregates. Results therefore indicate that the criterion of nuclear maturity used for donor oocyte selection does not always correspond to cytoplasmic maturity, which can partially explain implantation failures with the use of donor oocytes.

Primary Ciliary Dyskinesia in a Portuguese Bronchiectasis Outpatient Clinic.

Primary ciliary dyskinesia (PCD) is a rare hereditary condition characterized by decreased mucociliary clearance of the airways and a compromised reproductive system, resulting in male and female infertility. Several mutations with varied clinical and pathological features have been documented, making diagnosis a challenging process. The purpose of this study is to describe the clinical and pathological features of Portuguese patients with PCD and to examine their genetic variants. A retrospective observational analysis was conducted with patients who were being monitored at a bronchiectasis outpatient clinic in 2022 and had a confirmed or high-likelihood diagnosis of PCD. In total, 17 patients were included in the study, with 12 (66.7%) having PCD confirmed and 5 (29.4%) having a high-likelihood diagnosis. Furthermore, 12 patients were subjected to transmission electron microscopy (TEM), with 7 (58.3%) exhibiting one hallmark defect. Genetic test data was obtained for all 17 patients, with 7 of them (41.2%) displaying a pathogenic/likely pathogenic mutation in homozygosity. To summarize, PCD is an uncommon but significant hereditary illness with consequences regarding morbidity and mortality. Despite the lack of a specific treatment, it is critical to confirm the diagnosis with genetic testing in order to effectively manage the disease and its accompanying disorders.

The Klinefelter Syndrome and Testicular Sperm Retrieval Outcomes.

Klinefelter syndrome (KS), caused by the presence of an extra X chromosome, is the most prevalent chromosomal sexual anomaly, with an estimated incidence of 1:500/1000 per male live birth (karyotype 47,XXY). High stature, tiny testicles, small penis, gynecomastia, feminine body proportions and hair, visceral obesity, and testicular failure are all symptoms of KS. Endocrine (osteoporosis, obesity, diabetes), musculoskeletal, cardiovascular, autoimmune disorders, cancer, neurocognitive disabilities, and infertility are also outcomes of KS. Causal theories are discussed in addition to hormonal characteristics and testicular histology. The retrieval of spermatozoa from the testicles for subsequent use in assisted reproduction treatments is discussed in the final sections. Despite testicular atrophy, reproductive treatments allow excellent results, with rates of 40-60% of spermatozoa recovery, 60% of clinical pregnancy, and 50% of newborns. This is followed by a review on the predictive factors for successful sperm retrieval. The risks of passing on the genetic defect to children are also discussed. Although the risk is low (0.63%) when compared to the general population (0.5-1%), patients should be informed about embryo selection through pre-implantation genetic testing (avoids clinical termination of pregnancy). Finally, readers are directed to a number of reviews where they can enhance their understanding of comprehensive diagnosis, clinical care, and fertility preservation.

Cystic fibrosis and primary ciliary dyskinesia: Similarities and differences.

Cystic fibrosis (CF) and Primary ciliary dyskinesia (PCD) are both rare chronic diseases, inherited disorders associated with multiple complications, namely respiratory complications, due to impaired mucociliary clearance that affect severely patients' lives. Although both are classified as rare diseases, PCD has a much lower prevalence than CF, particularly among Caucasians. As a result, CF is well studied, better recognized by clinicians, and with some therapeutic approaches already available. Whereas PCD is still largely unknown, and thus the approach is based on consensus guidelines, expert opinion, and extrapolation from the larger evidence base available for patients with CF. Both diseases have some clinical similarities but are very different, necessitating different treatment by specialists who are familiar with the complexities of each disease.This review aims to provide an overview of the knowledge about the two diseases with a focus on the similarities and differences between both in terms of disease mechanisms, common clinical manifestations, genetics and the most relevant therapeutic options. We hoped to raise clinical awareness about PCD, what it is, how it differs from CF, and how much information is still lacking. Furthermore, this review emphasises the fact that both diseases require ongoing research to find better treatments and, in particular for PCD, to fill the medical and scientific gaps.

Further Insights on RNA Expression and Sperm Motility.

Asthenozoospermia is one of the main causes of male infertility and it is characterized by reduced sperm motility. Several mutations in genes that code for structural or functional constituents of the sperm have already been identified as known causes of asthenozoospermia. In contrast, the role of sperm RNA in regulating sperm motility is still not fully understood. Consequently, here we aim to contribute to the knowledge regarding the expression of sperm RNA, and ultimately, to provide further insights into its relationship with sperm motility. We investigated the expression of a group of mRNAs by using real-time PCR (CATSPER3, CFAP44, CRHR1, HIP1, IQCG KRT34, LRRC6, QRICH2, RSPH6A, SPATA33 and TEKT2) and the highest score corresponding to the target miRNA for each mRNA in asthenozoospermic and normozoospermic individuals. We observed a reduced expression of all mRNAs and miRNAs in asthenozoospermic patients compared to controls, with a more accentuated reduction in patients with progressive sperm motility lower than 15%. Our work provides further insights regarding the role of RNA in regulating sperm motility. Further studies are required to determine how these genes and their corresponding miRNA act regarding sperm motility, particularly KRT34 and CRHR1, which have not previously been seen to play a significant role in regulating sperm motility.

The Role of ROS as a Double-Edged Sword in (In)Fertility: The Impact of Cancer Treatment.

Tumor cells are highly resistant to oxidative stress resulting from the imbalance between high reactive oxygen species (ROS) production and insufficient antioxidant defenses. However, when intracellular levels of ROS rise beyond a certain threshold, largely above cancer cells' capacity to reduce it, they may ultimately lead to apoptosis or necrosis. This is, in fact, one of the molecular mechanisms of anticancer drugs, as most chemotherapeutic treatments alter redox homeostasis by further elevation of intracellular ROS levels or inhibition of antioxidant pathways. In traditional chemotherapy, it is widely accepted that most therapeutic effects are due to ROS-mediated cell damage, but in targeted therapies, ROS-mediated effects are mostly unknown and data are still emerging. The increasing effectiveness of anticancer treatments has raised new challenges, especially in the field of reproduction. With cancer patients' life expectancy increasing, many aiming to become parents will be confronted with the adverse effects of treatments. Consequently, concerns about the impact of anticancer therapies on reproductive capacity are of particular interest. In this review, we begin with a short introduction on anticancer therapies, then address ROS physiological/pathophysiological roles in both male and female reproductive systems, and finish with ROS-mediated adverse effects of anticancer treatments in reproduction.

Discordance between human sperm quality and telomere length following differential gradient separation/swim-up.

BACKGROUND: Strong evidence has suggested an important role of telomeres in meiosis, fertilization, and embryo development. PURPOSE: To determine if sperm telomere length (STL) in sperm purified by differential gradient centrifugation followed by swim-up (selected STL) is correlated with sperm quality and clinical outcomes. METHODS: Relative selected STL was assessed by quantitative polymerase chain reaction (Q-PCR) in 78 consecutive assisted reproductive technology (ART) treatments during 2017. Statistical analyses were performed in the totality of patients, and in normozoospermic and non-normozoospermic patients. These included correlations between selected STL and sperm quality parameters, embryological parameters (multivariable linear regression), and clinical parameters (multivariable logistic regression). RESULTS: No significant correlations were found between selected STL and sperm quality in the total population. However, selected STL was significantly correlated with total sperm count (r = 0.361; P = 0.039) and sperm DNA fragmentation-post-acrosomal region pattern (r = - 0.464; P = 0.030) in normozoospermic patients. No relation was observed between selected STL and clinical outcomes in any clinical group. CONCLUSIONS: As the correlations observed in normozoospermic patients were not representative of the whole heterogeneous population, differences in the sperm characteristics of the study population may lead to discrepant results when evaluating the association of STL with sperm quality. Since the total population selected STL was not related with sperm quality and with clinical outcomes, results do not support the use of selected STL measurement to evaluate the reproductive potential of the male patient or to predict the success rates of ART treatments.

Use of antioxidant could ameliorate the negative impact of etoposide on human sperm DNA during chemotherapy.

RESEARCH QUESTION: A previous study showed that N-acetylcysteine (NAC), used after in-vitro exposure to the gonadotoxic chemotherapeutic drug etoposide, has the ability to decrease DNA damage in human spermatozoa; however, it showed no benefit when used before exposure. This study aimed to evaluate the impact of the NAC on the preservation of sperm quality during in-vitro exposure to etoposide. DESIGN: Twenty semen samples were submitted to four experimental conditions: control, NAC-only incubation, etoposide-only incubation, and concomitant etoposide and NAC incubation. After in-vitro incubation, semen parameters, sperm chromatin condensation, sperm DNA fragmentation, sperm oxidative stress and sperm metabolism were used to evaluate the role of NAC in protecting human spermatozoa from etoposide. RESULTS: Etoposide did not affect semen parameters, nor did it cause sperm oxidative damage or alterations in glycolytic profile. However, it induced chromatin decondensation and DNA fragmentation, which were fully prevented by NAC. CONCLUSIONS: NAC was able to protect sperm DNA integrity during etoposide treatment in vitro, suggesting that NAC may be useful as an adjuvant agent in preserving male fertility during chemotherapy treatments.

Stereological study of organelle distribution in human oocytes at metaphase I.

We have previously presented a stereological analysis of organelle distribution in human prophase I oocytes. In the present study, using a similar stereological approach, we quantified the distribution of organelles in human metaphase I (MI) oocytes also retrieved after ovarian stimulation. Five MI oocytes were processed for transmission electron microscopy and a classical manual stereological technique based on point-counting with an adequate stereological grid was used. Kruskal-Wallis and Mann-Whitney U-tests with Bonferroni correction were used to compare the means of relative volumes (Vv) occupied by organelles. In all oocyte regions, the most abundant organelles were mitochondria and smooth endoplasmic reticulum (SER) elements. No significant differences were observed in Vv of mitochondria, dictyosomes, lysosomes, or SER small and medium vesicles, tubular aggregates and tubules. Significant differences were observed in other organelle distributions: cortical vesicles presented a higher Vv (P = 0.004) in the cortex than in the subcortex (0.96% vs 0.1%) or inner cytoplasm (0.96% vs 0.1%), vesicles with dense granular contents had a higher Vv (P = 0.005) in the cortex than in the subcortex (0.1% vs 0%), and SER large vesicles exhibited a higher Vv (P = 0.011) in the inner cytoplasm than in the subcortex (0.2% vs 0%). Future stereological analysis of metaphase II oocytes and a combined quantitative data of mature and immature oocytes, will enable a better understanding of oocyte organelle distribution during in vivo maturation. Combined with molecular approaches, this may help improve stimulation protocols and in vitro maturation methods.

Protective role of N-acetylcysteine (NAC) on human sperm exposed to etoposide.

BACKGROUND: Although recent progress in cancer treatment has increased patient survival and improved quality of life, reproductive side effects are still for concern. One way to decrease gonadal impairment is to use cytoprotectors. In testicular cancer, etoposide is generally used in combination with other agents, but there are no in-vitro studies of sperm exposure to etoposide and cytoprotectors, namely N-acetylcysteine (NAC). METHODS: Twenty semen samples were individually divided into five groups: control, incubation with NAC alone, incubation with etoposide alone, sequential exposure of NAC followed by etoposide (pre-treatment) and sequential exposure of etoposide followed by NAC (post-treatment). Sperm characteristics, chromatin condensation (aniline blue), DNA fragmentation (TUNEL), oxidative stress (OxyDNA labelling) and glutathione quantification were used to evaluate the capabilities of NAC as a prophylactic (pre-treatment) or ameliorator (post-treatment) agent over the effects caused in sperm during in-vitro exposure to etoposide. RESULTS: No deleterious effects were observed on sperm motility or sperm membrane integrity. Results revealed that prophylactic use of NAC (pre-treatment) increased rates of immature sperm chromatin as compared to ameliorator use of NAC (post-treatment), and increased rates of sperm DNA fragmentation in relation to controls. Pre and post-treatment with NAC increased oxidative levels in comparison to controls, but also increased levels of cellular antioxidant glutathione. CONCLUSIONS: The results indicate that NAC has the ability to counteract etoposide-induced toxicity rather than preventing the etoposide cytotoxic effects over sperm DNA, suggesting that the administration of NAC to cells formerly exposed to etoposide is preferable to its prophylactic use. As the results evidenced that NAC seems to be more efficient in attenuating sperm etoposide cytotoxic effects instead of being used as a chemoprophylactic agent, this reinforces the idea that there might be a new NAC mechanism over DNA.

CONTEXTE: Bien que les récents progrès dans le traitement des cancers aient augmenté la survie des patients et amélioré leur qualité de vie, les effets secondaires sur la reproduction restent encore des motifs d'inquiétude. Une façon de réduire les altérations gonadiques consiste en l'utilisation de cytoprotecteurs. Dans le cancer du testicule, l'étoposide est habituellement utilisée en association avec d'autres agents, mais il n'existe aucune étude in vitro de l'exposition des spermatozoïdes à l'étoposide et à des cytoprotecteurs, notamment la N-acétylcystéine (NAC). MATÉRIEL ET MÉTHODES: Vingt échantillons de sperme ont été répartis en cinq groupes : témoins, incubés avec NAC seule, incubés avec étoposide seul, exposés séquentiellement à NAC puis à étoposide (pré traitement), et exposés séquentiellement à étoposide puis à NAC (post traitement). Les paramètres spermatiques, la condensation de la chromatine (bleu d'aniline), la fragmentation de l'ADN (TUNEL), le stress oxydatif (marquage OxyDNA) et la quantification du glutathion ont été utilisés pour évaluer les capacités de NAC comme agent prophylactique (pré traitement) ou comme améliorateur (post traitement) des effets causés sur les spermatozoïdes lors d'une exposition in vitro à l'étoposide. RÉSULTATS: Aucun effet délétère n'a été observé sur la mobilité ou sur l'intégrité de la membrane des spermatozoïdes. Les résultats montrent que l'utilisation prophylactique (pré traitement) de NAC augmente les taux des spermatozoïdes avec chromatine immature en comparaison de l'utilisation amélioratrice (post traitement) de NAC, et augmente les taux de fragmentation de l'ADN des spermatozoïdes par rapport aux témoins. L'utilisation de NAC en pré et post traitement augmente les taux d'oxydation par rapport aux témoins, mais augmente aussi les taux de glutathion anti-oxydant cellulaire. CONCLUSIONS: Les résultats indiquent que NAC possède la capacité de contrebalancer la toxicité induite par l'étoposide plutôt que celle d'empêcher les effets cytotoxiques de l'étoposide sur l'ADN des spermatozoïdes ; ceci suggère que l'administration de NAC aux cellules préalablement exposées à l'étoposide est préférable à son utilisation prophylactique. Comme les résultats témoignent que NAC semble être plus efficace à atténuer les effets cytotoxiques de l'étoposide sur les spermatozoïdes plutôt que d'être utilisée comme agent chimio prophylactique, ceci renforce l'idée qu'il pourrait exister un nouveau mécanisme de NAC sur l'ADN. MOTS-CLÉS: N-acétylcystéine (NAC), Etoposide, Fragmentation de l'ADN spermatique, Stress oxydatif spermatique.

Structural and molecular analysis of the cancer prostate cell line PC3: Oocyte zona pellucida glycoproteins.

The human oocyte zona pellucida (ZP) is made of four glycoproteins, ZP1-ZP4. Recently, the prostate adenocarcinoma and prostate cancer PC3 cell-line were shown to express the human oocyte ZP3 glycoprotein, which was evaluated in a single report subject to patent. To further clarify whether oocyte zona pellucida glycoproteins are expressed in prostate cancer tissue and PC3-cells, in this report we evaluated protein expression of the four ZP glycoproteins in normal prostate tissue, prostate adenocarcinoma tissue and PC3-cells, and performed quantitative mRNA expression of the four ZP glycoproteins in the PC3 cell-line. Furthermore, as PC3-cells have not yet been studied in detail regarding their ultrastructural characteristics, in the present report we bring forward the detailed ultrastructure of PC3-cells. PC3-cells were divided into pavement and aggregated cells. We observed new ultrastructural features in pavement and aggregated cells, with the later exhibiting two different cell types. In prostate carcinoma tissue and PC3-cells we found protein expression of the four oocyte glycoproteins, ZP1, ZP2, ZP3 and ZP4. In addition, mRNA expression studies revealed expression of ZP1, ZP3 and ZP4 glycoproteins, but not of ZP2. Interestingly, the ZP1 mRNA product exhibited intron retention.

Major regulatory mechanisms involved in sperm motility.

The genetic bases and molecular mechanisms involved in the assembly and function of the flagellum components as well as in the regulation of the flagellar movement are not fully understood, especially in humans. There are several causes for sperm immotility, of which some can be avoided and corrected, whereas other are related to genetic defects and deserve full investigation to give a diagnosis to patients. This review was performed after an extensive literature search on the online databases PubMed, ScienceDirect, and Web of Science. Here, we review the involvement of regulatory pathways responsible for sperm motility, indicating possible causes for sperm immotility. These included the calcium pathway, the cAMP-dependent protein kinase pathway, the importance of kinases and phosphatases, the function of reactive oxygen species, and how the regulation of cell volume and osmolarity are also fundamental components. We then discuss main gene defects associated with specific morphological abnormalities. Finally, we slightly discuss some preventive and treatments approaches to avoid development of conditions that are associated with unspecified sperm immotility. We believe that in the near future, with the development of more powerful techniques, the genetic causes of sperm immotility and the regulatory mechanisms of sperm motility will be better understand, thus enabling to perform a full diagnosis and uncover new therapies.

Ultrastructural and cytogenetic analyses of mature human oocyte dysmorphisms with respect to clinical outcomes.

PURPOSE: The study aimed to describe the ultrastructure of two human mature oocyte intracytoplasmic dysmorphisms, the bull-eye inclusion and the granular vacuole, with evaluation of clinical outcomes after intracytoplasmic sperm injection (ICSI) treatment. METHODS: We retrospectively evaluated 4099 consecutive ICSI cycles during the period 2003-2013. Three groups were compared: controls, those with a bulls-eye inclusion, and those with granular vacuoles. Oocyte dysmorphisms were evaluated by transmission electron microscopy and in situ fluorescence hybridization (FISH). Detailed data on demographic and stimulation characteristics, as well as on embryological, clinical, and newborn outcomes, are fully presented. RESULTS: The bull-eye inclusion is a prominent smooth round structure containing trapped vesicles, being surrounded by lipid droplets. The presence of this dysmorphism in the oocyte cohort had no clinical impact except when transferred embryos were exclusively derived from dysmorphism oocytes. The granular vacuole is delimited by a discontinuous double membrane and contains lipid droplets and vesicles. As FISH analysis revealed the presence of chromosomes, they probably represent pyknotic nuclei. The presence of this dysmorphism in the oocyte cohort had no clinical impact except when at least one transferred embryo was derived from dimorphic oocytes. CONCLUSIONS: Poor clinical outcomes were observed with transfer of embryos derived from dysmorphism oocytes, although without causing gestation or newborn problems. The bull-eye inclusion and granular vacuoles may thus be new prognostic factors for clinical outcomes.

Emerging Potential of Natural Products as an Alternative Strategy to Pharmacological Agents Used Against Metabolic Disorders.

BACKGROUND: Human metabolism is an essential biological process that involves the consumption of different substrates to ensure the nutritional and energetic needs of cells. The disruption of this highly regulated system constitutes the onset of several disorders/dysfunctions such as diabetes mellitus, cardiovascular diseases and hypertension. OBJECTIVE: In this review, we propose to discuss promising natural products that can act as modulators of cell metabolism and point towards possible targets to take into account in the development of new therapies against metabolic diseases. METHODS: After having defined our main focus, we undertook an intensive search of bibliographic databases to select the peer-reviewed papers that fits within the review thematic. The information of the screened papers was described in an organized manner through the review and different types of studies were included. RESULTS: Two hundred and seventy papers were included in the review, as well as one reliable website from the World Health Organization. Several articles described that pharmacological agents are commonly used to counteract metabolic disorders. However, in many cases these products are insufficient, represent high costs to health care systems and are associated with several undesirable effects, highlighting the need to search for new therapies. Notably, many papers reported the promising results of natural products in the treatment of several metabolic disorders, constituting a possible alternative or complementary strategy to pharmacological agents. CONCLUSION: The findings of this review confirm that the currently available treatments for metabolic disorders and its associated complications remain far below the expected results.

Estradiol modulates Na(+) -dependent HCO3 (-) transporters altering intracellular pH and ion transport in human Sertoli cells: A role on male fertility?

BACKGROUND INFORMATION: Infertile men often present deregulation of serum estrogen levels. Notably, high levels of estradiol (E2) are associated with low sperm production and quality. Sertoli cells (SCs) are responsible for spermatogenesis maintenance and are major targets for the hormonal signalling that regulates this complex process. RESULTS: In this study, we used primary cultures of human SCs and studied the localisation, expression and functionality of the Na(+) -dependent HCO3 (-) transporters by confocal microscopy, immunoblot, epifluorescence and voltage clamp after 24 h of exposure to E2 (100 nM). All studied transporters were identified in human SCs. In E2-treated human SCs, there was an increase in NBCn1, NBCe1 and NDCBE protein levels, as well as an increase in intracellular pH and a decrease in transcellular transport. CONCLUSIONS: We report an association between increased levels of E2 and the expression/function of Na(+) -dependent HCO3 (-) transporters in human SCs. Our results provide new evidence on the mechanisms by which E2 can regulate SCs physiology and consequently spermatogenesis. These mechanisms may have an influence on male reproductive potential and help to explain male infertility conditions associated with estrogen deregulation. SIGNIFICANCE: Exposure to E2 increased human SCs intracellular pH. E2 is a modulator of ionic transcellular transport in human SCs.

A stereological study on organelle distribution in human oocytes at prophase I.

The ultrastructural analysis of human oocytes at different maturation stages has only been descriptive. The aim of this study was to use a stereological approach to quantify the distribution of organelles in oocytes at prophase I (GV). Seven immature GV oocytes were processed for transmission electron microscopy and a classical manual stereological technique based on point-counting with an adequate stereological grid was used. The Kruskal-Wallis test and Mann-Whitney U-test with Bonferroni correction were used to compare the means of the relative volumes occupied by organelles in oocyte regions: cortex (C), subcortex (SC) and inner cytoplasm (IC). Here we first describe in GV oocytes very large vesicles of the smooth endoplasmic reticulum (SER), vesicles containing zona pellucida-like materials and coated vesicles. The most abundant organelles were the very large vesicles of the SER (6.9%), mitochondria (6.3%) and other SER vesicles (6.1%). Significant differences in organelle distribution were observed between ooplasm regions: cortical vesicles (C: 1.3% versus SC: 0.1%, IC: 0.1%, P = 0.001) and medium-sized vesicles containing zona pellucida-like materials (C: 0.2% versus SC: 0.02%, IC: 0%, P = 0.004) were mostly observed at the oocyte cortex, whereas mitochondria (C: 3.6% versus SC: 6.0%, IC: 7.2%, P = 0.005) were preferentially located in the subcortex and inner cytoplasm, and SER very large vesicles (IC: 10.1% versus C: 0.9%, SC: 1.67%, P = 0.001) in the oocyte inner cytoplasm. Further quantitative studies are needed in immature metaphase-I and mature metaphase-II oocytes, as well as analysis of correlations between ultrastructural and molecular data, to better understand human oocyte in vitro maturation.

Sperm DNA fragmentation is related to sperm morphological staining patterns.

In this prospective comparative study, sperm DNA fragmentation (sDNAfrag) was compared at each step of a sequential semen preparation, with semen parameters according to their degree of severity. At each step (fractions) of the sequential procedure, sDNAfrag was determined: fresh (Raw), after gradient centrifugation, washing, and swim-up (SU) for 70 infertile men enrolled in intracytoplasmic sperm injection cycles. sDNAfrag significantly (P = 0.04; P < 0.0001) decreased throughout the steps of semen preparation, with centrifugation and washing not increasing it. A negative correlation to sperm motility was observed in Raw and SU fractions, and a higher sDNAfrag was observed in samples with lower semen quality. Our results confirm that the steps of the sequential procedure do not compromise sperm DNA integrity and progressively decreased sDNAfrag regardless of the sperm abnormality and that semen parameters with lower quality present higher sDNAfrag. Four distinct patterns were observed, of which the entire sperm head staining was the pattern most expressed in all studied fractions. Additionally, the sperm head gene-rich region staining pattern was reduced by the procedure. This suggests that pattern quantification might be a useful adjunct when performing sDNAfrag testing for male infertility.