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.

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.

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.

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.

Leptin modulates human Sertoli cells acetate production and glycolytic profile: a novel mechanism of obesity-induced male infertility?

Human feeding behavior and lifestyle are gradually being altered, favoring the development of metabolic diseases, particularly type 2 diabetes and obesity. Leptin is produced by the adipose tissue acting as a satiety signal. Its levels have been positively correlated with fat mass and hyperleptinemia has been proposed to negatively affect male reproductive function. Nevertheless, the molecular mechanisms by which this hormone affects male fertility remain unknown. Herein, we hypothesize that leptin acts on human Sertoli cells (hSCs), the "nurse cells" of spermatogenesis, altering their metabolism. To test our hypothesis, hSCs were cultured without or with leptin (5, 25 and 50ng/mL). Leptin receptor was identified by qPCR and Western blot. Protein levels of glucose transporters (GLUT1, GLUT2 and GLUT3), phosphofructokinase, lactate dehydrogenase (LDH) and monocarboxylate transporter 4 (MCT4) were determined by Western Blot. LDH activity was assessed and metabolite production/consumption determined by proton nuclear magnetic resonance. Oxidative damage was evaluated by assessing lipid peroxidation, protein carbonilation and nitration. Our data shows that leptin receptor is expressed in hSCs. The concentration of leptin found in lean, healthy patients, upregulated GLUT2 protein levels and concentrations of leptin found in lean and obese patients increased LDH activity. Of note, all leptin concentrations decreased hSCs acetate production illustrating a novel mechanism for this hormone action. Moreover, our data shows that leptin does not induce or protect hSCs from oxidative damage. We report that this hormone modulates the nutritional support of spermatogenesis, illustrating a novel mechanism that may be linked to obesity-induced male infertility.

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.

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.

Estrogenic regulation of bicarbonate transporters from SLC4 family in rat Sertoli cells.

The formation of competent spermatozoa is a complex event that depends on the establishment of adequate environments throughout the male reproductive tract. Bicarbonate is essential not only to ionic homeostasis but also to pH maintenance along the male reproductive tract. Previous studies support an association of high 17ß-estradiol (E2) levels with modulation of specific ion transporters expression. Herein we determined the effect of E2 on the expression/functionality of SLC4 family bicarbonate transporters in rat Sertoli cells (SCs). All studied transporters [anion exchanger 2 (AE2), Na(+)-driven Cl(-)/HCO3 (-) exchanger (NDCBE), electrogenic Na(+)/HCO3 (-) co-transporters (NBCe1), and electroneutral Na(+)/HCO3 (-) co-transporters (NBCn1)] were identified in SCs, being AE2 and NBCn1 the most abundant. In E2-treated cells (100 nM), increases in AE2 and NBCn1 protein levels were observed, as well as altered transcellular transport. E2-treated SCs presented a significant perturbation of ATP-induced short-circuit current. This alteration was concurrent with augmented AE2 and NBCn1 levels. Overall, we report a relation between increased E2 levels and the expression/function of AE2 and NBCn1 in rat SCs, providing new evidence on the mechanisms by which E2 can regulate SCs physiology and consequently spermatogenesis, with direct influence on male reproductive potential.

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.

Ultrastructural analysis of five patients with total sperm immotility.

Asthenozoospermia has been related to structural defects of the sperm flagellum. However, few reports have studied in detail the ultrastructure of sperm with total immotility. We present an ultrastructural study of sperm from five patients with total sperm immotility, four due to dysplasia of the fibrous sheath (DFS) and one with situs-inversus. Of the four patients with DFS, three cases presented a hypertrophic and hyperplastic fibrous sheath that invaded the midpiece space, absence of the annulus, and a short midpiece containing a few disorganized and pale mitochondria. Of these cases, two presented absence of the central complex and radial spokes; another additionally presented absence of dynein arms and nexin bridges; and the other patient presented an intact annulus with a dysplastic fibrous sheath restricted to the principal piece with disorganized microtubule doublets. The patient with situs-inversus presented severe respiratory symptoms, with absence of dynein arms and nexin bridges. In conclusion, we present three cases with DFS associated with total sperm immotility, abnormal mitochondria, and absence of the annulus, central pair complex and radial spokes, of which one had in addition absence of dynein arms and nexin bridges. We also describe a patient, with total sperm immotility and a different presentation of DFS, as the annulus was present and the dysplastic fibrous sheath was restricted to the principal piece. These findings thus confirm the heterogeneity of the DFS condition. The changes observed in the patient with situs-inversus also further support previous observations.

DNA fragmentation in human sperm after magnetic-activated cell sorting.

PURPOSE: As fertilization with unselected apoptotic spermatozoa may contribute to failures in assisted reproductive techniques, it has become essential to remove this type of sperm in order to increase the success rates. Magnetic-activated cell sorting (MACS) is a sperm preparation technique that isolates non-apoptotic spermatozoa based on the expression of phosphatidylserine in the membrane of apoptotic sperm. Therefore, we aimed to evaluate whether there was a significant decrease in sperm DNA fragmentation (sDNAfrag) and verify which protocol was the most efficient. METHODS: Hundred semen samples were allocated into five distinct groups and processed according to a combination of MACS with density gradient centrifugation (DGC) and swim-up (SU) techniques. Sperm DNA fragmentation was evaluated by terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay. RESULTS: Groups DGC-SU (73.4 %), DGC-MACS-SU (78.9 %), DGC-SU-MACS (53.8 %) and MACS-SU (73.5 %) presented a significant decrease in sDNAfrag but the highest reduction rate was obtained with MACS-DGC-SU (83.3 %). The later was also negatively correlated with sperm vitality, membrane integrity and progressive motility. Additionally, teratozoospermic patients presented a tendency to have lower sDNAfrag reduction rates than asthenozoospermic and asthenoteratozoospermic patients. CONCLUSIONS: Based on the results, MACS showed potential to optimize the sDNAfrag reduction rate, when applied to raw semen, before DGC and SU, especially in samples with low values of progressive motility, vitality and hypoosmotic swelling test.

Aquaporin-4 as a molecular partner of cystic fibrosis transmembrane conductance regulator in rat Sertoli cells.

Sertoli cells (SCs) form the blood-testis barrier (BTB) that controls the microenvironment where the germ cells develop. The cystic fibrosis transmembrane conductance regulator (CFTR) plays an essential role to male fertility and it was recently suggested that it may promote water transport. Interestingly, Aquaporin-4 (AQP4) is widely expressed in blood barriers, but was never identified in SCs. Herein we hypothesized that SCs express CFTR and AQP4 and that they can physically interact. Primary SCs cultures from 20-day-old rats were maintained and CFTR and AQP4 mRNA and protein expression was assessed by RT-PCR and Western blot, respectively. The possible physical interaction between CFTR and AQP4 was studied by co-immunoprecipitation. We were able to confirm the presence of CFTR at mRNA and protein level in cultured rat SCs. AQP4 mRNA analysis showed that cultured rat SCs express the transcript variant c of AQP4, which was followed by immunodetection of the correspondent protein. The co-immunoprecipitation experiments showed a direct interaction between AQP4 and CFTR in cultured rat SCs. Our results suggest that CFTR physically interacts with AQP4 in rat SCs evidencing a possible mechanism by which CFTR can control water transport through BTB. The full enlightenment of this particular relation between CFTR and AQP4 may point towards possible therapeutic targets to counteract male subfertility/infertility in men with Cystic Fibrosis and mutations in CFTR gene, which are known to impair spermatogenesis due to defective water transport.

Physiology of na+/h+ exchangers in the male reproductive tract: relevance for male fertility.

The maintenance of pH homeostasis in the male reproductive tract is kept through the involvement of several mechanisms, among which is included the transmembranous movement of H(+) ions. Na(+)-H(+) exchangers (SLC9, solute carrier 9 family members) are among the membrane transporters known to participate in intracellular and extracellular pH regulation but also have important roles in salt and water absorption across epithelia and in the regulation of cell volume. The presence of several Na(+)-H(+) exchangers has been reported in the male reproductive tract. Their involvement in the processes that ensure the correct pursuance of the spermatogenetic event and spermatozoa maturation has been suggested. Indeed, the formation of mature spermatozoa is highly dependent on the maintenance of adequate ductal luminal milieu pH and ionic balance. Perturbations in these processes result in reduced male reproductive potential and consequently male subfertility and/or infertility. Thus, it is imperative to understand H(+) transport dynamics in order to identify and counteract possible alterations associated with reduced male fertility caused by pathological conditions. Herein, we will discuss the expression pattern and physiological roles of SLC9 family members in the cells of the male reproductive tract as well as the molecular basis of H(+) transport and its involvement in male reproductive potential.

Aquaporin-9 is expressed in rat Sertoli cells and interacts with the cystic fibrosis transmembrane conductance regulator.

Men with mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene are usually subfertile/infertile. Besides playing a role in Cl(-)/HCO3(-) transport, it has been proposed that CFTR interacts with water membrane transport systems, particularly aquaporins, to control seminiferous tubular secretion, which is regulated by the somatic Sertoli cells (SCs). As aquaporin-9 (AQP9) is highly expressed throughout the male reproductive tract, we hypothesized that it is also present in rat SCs and that it physically interacts with CFTR. To test this hypothesis, primary cultures of rat SCs were established, and expression of CFTR and AQP9 was assessed by RT-polymerase chain reactions (mRNA) and Western blot analysis (protein). A coimmunoprecipitation assay was used to evaluate the physical interaction between CFTR and AQP9. Our results show that CFTR and AQP9 are expressed in rat SCs. We were also able to detect a molecular interaction between CFTR and AQP9 in rat SCs. This is the first report describing the presence of AQP9, and its interaction with CFTR, in rat SCs. Moreover, our results provide evidence that CFTR is involved in water homeostasis of the seminiferous tubular secretion. These mechanisms may open new insights on therapeutic targets to counteract subfertility/infertility in men with cystic fibrosis and mutations in the CFTR gene.

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.

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.

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.