Pharmacological administration of recombinant human AMH rescues ovarian reserve and preserves fertility in a mouse model of chemotherapy, without interfering with anti-tumoural effects.

PURPOSE: To determine whether pharmacological administration of recombinant human anti-Mullerian hormone (rAMH) protects the ovarian reserve and preserves fertility without interfering with anti-tumoural cytotoxic action of chemotherapy. METHODS: Intraperitoneal delivery of rAMH and ovarian post-receptor activity were assessed with immunohistochemistry and western blot. Differential follicle counts and reproductive outcomes were assessed after cyclophosphamide (Cy) administration, with/without concurrent administration of rAMH. Interference of rAMH with Cy chemotoxicity was assessed on a human breast cancer cell line and an in vivo mouse model of human leukaemia. RESULTS: rAMH reached the ovary after intraperitoneal injection and demonstrated post-receptor bioactivity. Cy administration in mice caused primordial follicle activation, as shown by a decrease in primordial follicle population accompanied by an increase in early growing follicles and granulosa cell proliferation. Co-administration of rAMH reduced follicle activation, thereby protecting the primordial follicle reserve, and improving long-term fertility and reproductive outcomes. rAMH co-administration did not interfere with the cytotoxic actions of Cy in vitro on breast cancer cell line or in vivo in a model of human leukaemia. CONCLUSION: This study demonstrates that rAMH is bioactive in the ovary for a limited time, and that pharmacological administration of rAMH during chemotherapy treatment reduces follicle activation and primordial follicle loss and significantly improves reproductive outcomes in a mouse model, and does not interfere with the therapeutic actions of the treatment. Further investigation is necessary to determine whether it has similar protective effects in the human ovary.

Regulatory T cells and Toll-like receptor 2 and 4 mRNA expression in infants with colic treated with Lactobacillus reuteri DSM17938.

Regulatory T cells induce immune homeostasis and the expression of Toll like receptors (TLRs); subsequent inflammatory cytokine release may be involved. Recent studies have shown a microbial imbalance in the gut of colicky infants (with a prevalence of gram-negative bacteria, such as Escherichia coli), and accumulating evidence has shown the efficacy of a probiotic (Lactobacillus reuteri) in breastfed subjects, but the underlying mechanism remains undefined. The study enrolled 59 infants younger than 60 days, of whom 34 subjects had colic and 25 were healthy controls. With a double-blind, placebo-controlled randomised study performed in our unit from October 2016 to July 2017, infants with colic were randomly assigned to receive oral daily L. reuteri DSM17938 (1×108 cfu) or placebo for 28 days. Peripheral blood was collected to assess the expression of FoxP3, TLR2 and TLR4 mRNA using real-time TaqMan RT-PCR at baseline and after the study period. Our findings showed increased mRNA expression of the transcription factor forkhead box P3 (FoxP3) in infants treated with L. reuteri DSM 17938 for 28 days (P<0.009) and increased TLR2 and TLR4 mRNA expression in both treated and placebo subjects. After L. reuteri administration for 28 days in infants with colic, we observed a significant decrease in daily crying time (302.3±19.86 min/day on day 0 vs 76.75±22.15 min/day on day 28, P=0.001). This study provides evidence that the observed increase in FoxP3 expression and reduction in crying time might be responses to probiotic treatment, while the increase in TLR2 and TLR4 mRNA expression might be related to age. Exploiting these new findings may lead to an unprecedented level of therapeutic control over immune tolerance using probiotics.

The histone deacetylase inhibitor givinostat (ITF2357) exhibits potent anti-tumor activity against CRLF2-rearranged BCP-ALL.

Leukemias bearing CRLF2 and JAK2 gene alterations are characterized by aberrant JAK/STAT signaling and poor prognosis. The HDAC inhibitor givinostat/ITF2357 has been shown to exert anti-neoplastic activity against both systemic juvenile idiopathic arthritis and myeloproliferative neoplasms through inhibition of the JAK/STAT pathway. These findings led us to hypothesize that givinostat might also act against CRLF2-rearranged BCP-ALL, which lack effective therapies. Here, we found that givinostat inhibited proliferation and induced apoptosis of BCP-ALL CRLF2-rearranged cell lines, positive for exon 16 JAK2 mutations. Likewise, givinostat killed primary cells, but not their normal hematopoietic counterparts, from patients carrying CRLF2 rearrangements. At low doses, givinostat downregulated the expression of genes belonging to the JAK/STAT pathway and inhibited STAT5 phosphorylation. In vivo, givinostat significantly reduced engraftment of human blasts in patient-derived xenograft models of CRLF2-positive BCP-ALL. Importantly, givinostat killed ruxolitinib-resistant cells and potentiated the effect of current chemotherapy. Thus, givinostat in combination with conventional chemotherapy may represent an effective therapeutic option for these difficult-to-treat subsets of ALL. Lastly, the selective killing of cancer cells by givinostat may allow the design of reduced intensity regimens in CRLF2-rearranged Down syndrome-associated BCP-ALL patients with an overall benefit in terms of both toxicity and related complications.

Semi-basements used as dwellings: hygienic considerations and analysis of the regulations.

Current housing shortage in Italy is forcing a growing number of individuals to use as living environment spaces that were originally devoted to other purposes. Among such spaces, semi-basements hold a particular relevance because of their specific characteristics and their effects on human health. The authors analyse the relatively scarce legislation about this topic at both national and regional level. The local Building Codes of the ten most populous cities of Italy are reviewed, assessing whether the use of semi-basements as living spaces is allowed and, if so, which restrictions and requirements are imposed. The authors conclude that, on one hand, further research is strongly needed to estimate the amount of exposed population and their health risk, on the other the existing legislation on the topic is often discretionary and deeply unhomogeneous across the country.

[Assessment of the hygienic and sanitary conditions of housing in a sample in Chieti (central Italy)]. / Analisi delle condizioni igienico-sanitarie di un campione di abitazioni del Comune di Chieti.

The study describes the housing conditions of 106 dwellings in the town of Chieti (Abruzzo region, central Italy), inspected by Local Health Authority, in the period between the 1st of January 2009 and the 28th of February 2011. In 59.4% of them lived Italians, while in the remaining 40.6% lived foreigners. 35.8% of the houses met minimum law requirements, 24.5% were anti-hygienic, 24.5% anti-hygienic and overcrowded, 13.2% overcrowded, 1.9% uninhabitable. The main issues were moisture and molds (52.8%). The second one was the inadequacy of floor area in relation with inhabitants (37.7%). Other problems were bedrooms (9.4%), baths (8.5%) and kitchens not complying minimum law requirements, but also not sufficient both ceiling hights (3.7%) and windows (2.8%) of rooms. Considering that human population in richest countries spend up to 90% of their lives in indoor environment, especially at home, the links between housing and health are really strong, and more must be done to improve general consideration of these themes both in general population and involved professionists (i.e. physicians, other health professionists, social workers, architects and engineers).

Bone maturity and thyroidal status at birth: role of the ultrasonographic evaluation of the distal femoral epiphysis.

PURPOSE: Thyroidal hormones are important for bone development, and ultrasonographic (US) evaluation of the distal femoral epiphysis (DFE) has recently been suggested as a new method for the assessment of skeletal maturity in infants. A delayed bone maturation, expressed by a smaller or absent DFE nucleus (in terms of DFE surface area, sum of the epiphyseal diameters, or DFE height and acetabular size), has been largely demonstrated in diagnosed hypothyroid infants, while no data analyze the role and meaning of the DFE dimensions (in terms of volume) in newborn before knowledge of the congenital neonatal screening results. The aims of the present study were to ultrasonographically evaluate the volume of DFE in newborns, and to determine whether it has any predictive role for the thyroidal status at birth. MATERIALS AND METHODS: 238 newborns (M/F: 121 /117) were evaluated. The gestational age, body weight and length at birth were registered. Neonatal screening for congenital hypothyroidism (CH), based on TSH levels on blood spot, was performed in all on the 3 rd day of life. The DFE volume was ultrasonographically evaluated, taking into account the three diameters of the DFE nucleus, within 48 hours of birth. RESULTS: No newborn was found to have CH on neonatal screening. The DFE volume ranged between 0.00 cm and 0.61 cm (mean 0.14 ± 0.10 cm, median 0.13 cm). The DFE volume did not differ between males and females, while it was significantly greater in at term babies than in preterm babies. No differences in TSH values at screening were noted among the groups. The DFE volume was significantly related to gestational age, birth weight and length. A significant relationship was found between the DFE volume and TSH concentrations at screening. CONCLUSION: US evaluation of DFE volume provides a simple method for assessing bone maturity at birth, which is related to gestational age, body weight and length at birth. Nonetheless, it has any predictive role for thyroidal status at birth, being not related to TSH levels on neonatal screening for CH.

Role of ultrasonography in the diagnosis and follow-up of pediatric eosinophilic gastroenteritis: a case report and review of the literature.

Eosinophilic gastroenteritis (EG) is a rare disease characterized by the infiltration of one or more layers of the digestive tract by eosinophilic leukocytes. The diagnosis is confirmed by histological examination of a characteristic biopsy, but radiological features are useful for diagnostic suspicion. We report the case of an adolescent boy with recurrent epigastric pain, nausea and vomiting, in whom sonographic features and eosinophilia of the peripheral blood suggested the diagnosis of EG. Moreover, we reviewed the radiological features of EG with particular regard to the role of sonography in the diagnosis and follow-up of EG, especially in children. We emphasize the utility of sonography in pediatric patients presenting with gastrointestinal symptoms, since it may provide useful information in a quick, inexpensive and noninvasive way. Ultrasonographic detection of features such as bowel wall thickness, ascites and peritoneal nodules may be largely suggestive of EG and may prevent other invasive exams and abdominal surgery. Ultrasonography can also be easily used in the follow-up of these patients, and may obviate the frequent and potentially dangerous exposure to radiation.

Ciprofloxacin-modified electrosynthesized hydrogel coatings to prevent titanium-implant-associated infections.

New promising and versatile materials for the development of in situ sustained release systems consisting of thin films of either poly(2-hydroxyethyl methacrylate) or a copolymer based on poly(ethylene-glycol diacrylate) and acrylic acid were investigated. These polymers were electrosynthesized directly on titanium substrates and loaded with ciprofloxacin (CIP) either during or after the synthesis step. X-ray photoelectron spectroscopy was used to check the CIP entrapment efficiency as well as its surface availability in the hydrogel films, while high-performance liquid chromatography was employed to assess the release property of the films and to quantify the amount of CIP released by the coatings. These systems were then tested to evaluate the in vitro inhibition of methicillin-resistant Staphylococcus aureus (MRSA) growth. Moreover, a model equation is proposed which can easily correlate the diameter of the inhibition haloes with the amount of antibiotic released. Finally, MG63 human osteoblast-like cells were employed to assess the biocompatibility of CIP-modified hydrogel coatings.

Epidural blood patch in Trendelenburg position pre-medicated with acetazolamide to treat spontaneous intracranial hypotension.

BACKGROUND: Spontaneous intracranial hypotension (SIH) is characterized by orthostatic headache, diffuse pachymeningeal enhancement on brain magnetic resonance imaging (MRI) and low cerebrospinal fluid (CSF) pressure. Treatment ranges from conservative management, such as bed rest, overhydration and caffeine, to invasive procedures, such as the autologous epidural blood patch (EBP), computed tomography (CT)-guided fibrin glue injection at the site of the leak and open surgical intervention. EBP has emerged as the treatment of choice for SIH when initial conservative measures fail to bring relief. METHODS: Forty-two patients with SIH were treated with lumbar autologous EBP in Trendelenburg position preceded by pre-medication with acetazolamide. RESULTS: A complete recovery was obtained in all patients after one (90%), two (5%) or three (5%) EBPs. After EBP, two patients (5%) also performed evacuation of bilateral chronic subdural hematoma with mass effect. CONCLUSIONS: Spontaneous intracranial hypotension can be effectively cured by lumbar autologous EBP in Trendelenburg position pre-medicated with acetazolamide.

Influenza vaccine effectiveness for the elderly: a cohort study involving general practitioners from Abruzzo, Italy.

INTRODUCTION: In all Italian regions influenza vaccine is routinely administered to the elderly population. However, vaccination impact has been rarely evaluated because of the high costs of conventional cohort investigations. A promising low-cost alternative approach uses administrative discharge data to derive vaccine effectiveness indicators (hospitalizations and/or deaths) and involves General Practitioners (GPs) to document the exposure. We conducted a cohort analysis using such approach to assess influenza vaccine effectiveness and to investigate the feasibility and validity of that methodology for routine vaccine evaluation. METHODS: During October 2006, all GPs from two Local Health Units (LHUs) were requested to indicate immunization status of all their patients in a specific form containing patient's demographic records. Immunization status information were also collected from Prevention Departments. Main outcomes were hospitalizations for influenza and/or pneumonia. Analyses were based upon random-effect logistic regression. RESULTS: Of a total of 414 GPs assisting 103,162 elderly, 116 GPs (28%) provided data on 32,457 individuals (31.5%). The sample was representative and had an overall 66.2% vaccina-tion rate. During the first semester 2007, the hospitalization rate was low in the sample, with only 7 elderly patients admitted for influenza and 135 for pneumonia. At either bivariate or multivariate analysis, vaccination did not significantly reduce the risk of in-hospital death, influenza or pneumonia admission. DISCUSSION: The study had minimal costs, recruited a large and representative sample size, and had no evidence of a substantial selection bias. Administrative and GP's data may be successively pooled to provide routine assessment of vaccination effectiveness.

Imaging spectrum of EBV-infection in a young patient.

A wide variety of atypical presentations with complications affecting multiple organ systems during acute infectious mononucleosis (IM) is described in the literature, with an increase in the number of teenagers who are susceptible to a severe case of the disease. We report a case of a 14-year-old girl with severe IM and acute abdominal pain. Ultrasonographic (US) evaluation showed a marked thickening of the gallbladder wall (GBW) with enlargement of some mesenteric lymph nodes. CT scan showed multiple enlarged lung nodules of various sizes and a small pleural and pericardial effusion; a hypodense solid mass of unknown etiology was detected in the anterior mediastinum, mimicking a malignant tumor. Hematological analysis of peripheral blood smear was performed to exclude neoplastic pathology. IM was identified as the only underlying disease. The patient was carefully monitored: clinical evaluation, laboratory analysis and US examination were repeated at weekly intervals, until recovery.

Water treatment in domiciliary hemodialysis equipment: ultraviolet irradiation and Sartobind membrane.

Water is a fundamental element in any hemodialysis device. It must be safe, free not only of micro-organisms but also of any organic or inorganic chemical contaminant. Up to now ion exchanger, reverse osmosis, activate carbon and chlorine are generally used for this purpose. The results are not satisfactory. Microbial contaminants survive and at some points multiply. Pyrogens and endotoxins are not completely eliminated. Chlorine itself adds more risk factors because of the compounds deriving from its interaction with organic molecules. A system to obtain safe water in hemodialysis equipment is described. It is based on the use of ultraviolet irradiation to eliminate any microbial contaminant and on Sartobind membranes to eliminate pyrogens, endotoxins, proteins and other unwanted molecules. Mobile domiciliary hemodialysis equipment was used. Ultraviolet lamps (30 mW/cm2) were applied at two points: after the ion exchange and after the reverse osmosis. Three Q100 Sartobind membranes were positioned immediately before the monitor. The values of cfu/mL counted in the water after the ion exchange ranged from 450 to 1,990, whereas before the monitor they dropped from 0.01 to 0.09 cfu/mL. The LAL test, positive before the Sartobind membrane, was negative thereafter. The system proposed has shown to be effective in guaranteeing safe water free of micro-organisms and endotoxins.

Thunderclap headache caused by spontaneous intracranial hypotension.

We report a group of 4 patients with thunderclap headache as the initial manifestation of spontaneous intracranial hypotension.

Spontaneous cerebrospinal fluid leak syndrome: report of 18 cases.

We examined a group of 18 consecutive patients with spontaneous cerebrospinal fluid leak syndrome (SCSFLS) and investigated clinical, MRI, radioisotope findings and therapeutic outcome of this syndrome.

Clonal spread of Acinetobacter baumannii in a general intensive care unit.

The epidemiological characterization of multiply resistant Acinetobacter baumannii isolates from a six-bed Intensive Care Unit (ICU) is described. Investigations for A. baumannii were performed in three subsequent surveillance studies. In the first study, surveillance cultures were taken from patients, health care personnel and the environment; in the second study surveillance cultures were taken at 0, 4, and 7 days from all patients admitted consecutively to the ward; and in the third study surveillance cultures were taken from patients, health care personnel and the environment. During the first study all four hospitalized patients were found to harbour A. baumannii. Hand cultures did not grow any A. baumannii when staff entered the ward from home, but 7 positive health care workers were identified out of 25 samples taken during work, and two cultures of environmental specimens grew A. baumannii. During the second study, 4 of 86 (4.6%) patients resulted colonized with A. baumannii. In the third epidemiological study, no A. baumannii was cultured from either patients, health care personnel or the environment. All isolates recovered from various patients or sources produced conserved macrorestriction Pulsed-Field Gel Electrophoresis (PFGE) patterns and showed the same antibiotic resistance; therefore, they can be considered indistinguishable. The same antibiotic resistance and macrorestriction patterns were observed in previously isolated A. baumannii strains in the ward during May 1997, suggesting the persistence of a single A. baumannii in the ICU. The present study confirms that molecular typing is an essential tool in the epidemiology and control of nosocomial infections, showing here the persistence of a single A. baumannii clone in the ICU. The origin of this strain remains unknown but, when basic infection control measures were reinforced, emphasizing the importance of hand antisepsis and judicious use of gloves, control of A. baumannii spread in the ward was achieved.

[Microbial air monitoring in operating theatre: active and passive samplings]. / Confronto tra due metodi di campionamento nel monitoraggio microbiologico in sala operatoria: campionamento attivo e passivo.

Microbial air contamination was evaluated in 11 operating theatres using active and passive samplings. SAS (Surface Air System) air sampling was used to evaluate cfu/m3 and settle plates were used to measure the index of microbial air contamination (IMA). Samplings were performed at the same time on three different days, at three different times (before, during and after the surgical activity). Two points were monitored (patient area and perimeter of the operating theatre). Moreover, the cfu/m3 were evaluated at the air inlet of the conditioner system. 74.7% of samplings performed at the air inlet and 66.7% of the samplings performed at the patient area before the beginning of the surgical activity (at rest) exceeded the 35 cfu/m3 used as threshold value. 100% of IMA values exceeded the threshold value of 5. Using both active and passive sampling, the microbial contamination was shown to increase significantly during activity. The cfu values were higher at the patient area than at the perimeter of the operating theatre. Mean values of the cfu/m3 during activity at the patient area ranged from a minimum of 61+/-41 cfu/m3 to a maximum of 242+/-136 cfu/m3; IMA values ranged from a minimum of 19+/-10 to a maximum of 129+/-60. 15.2% of samplings performed at the patient area using SAS and 75.8% of samplings performed using settle plates exceeded the threshold values of 180 cfu/m3 and 25 respectively, with a significant difference of the percentages. The highest values were found in the operating theatre with inadequate structural and managerial conditions. These findings confirm that the microbiological quality of air may be considered a mirror of the hygienic conditions of the operating theatre. Settle plates proved to be more sensitive in detecting the increase of microbial air contamination related to conditions that could compromise the quality of the air in operating theatres.

Postural headache in a patient with Marfan's syndrome.

A 26-year-old man with Marfan's syndrome had postural headache. Brain MRI with gadolinium showed diffuse pachymeningeal enhancement. MRI myelography revealed bilateral multiple large meningeal diverticula at sacral nerve roots level. He was suspected to have spontaneous intracranial hypotension syndrome. Eight days later headache improved with bed rest and hydration. One month after the onset he was asymptomatic and 3 months later brain MRI showed no evidence of diffuse pachymeningeal enhancement. The 1-year follow-up revealed no neurological abnormalities. The intracranial hypotension syndrome likely resulted from a CSF leak from one of the meningeal diverticula. In conclusion patients with spinal meningeal diverticula (frequently seen in Marfan's syndrome) might be at increased risk of developing CSF leaks, possibly secondary to Valsalva maneuver or minor unrecognized trauma.

Lack of influence of body exhaust gowns on aerobic bacterial surface counts in a mixed-ventilation operating theatre. A study of 62 hip arthroplasties.

Aerobic bacterial surface contamination was studied with and without the use of body exhaust gowns in an operating room equipped with mixed/turbulent ventilation and separate operating and anaesthetic areas during 62 hip joint arthroplasties. In 31 operations conventional gowns were used, and 31 were performed with body exhaust gowns. Bacterial surface contamination was monitored in the operating and anaesthetic area using 9 cm diameter settle plates (1+1) and nitrocellulose membranes (2+2) transferred after sampling to nutrient pads. Compared with conventional clothing, the use of body exhaust gowns did not significantly reduce the microbial contamination (P=0.1-0.7). On the settle plates 1 m from the patient 279+/-326 cfu/m(2)/h were observed with conventional clothing compared with 142+/-227 cfu/m(2)/h with body exhaust gowns. The first membrane located on the patient in the sterile area detected 250+/-590 cfu/m(2)/h with conventional clothing and 210+/-320 cfu/m(2)/h with exhaust gowns. For the second membrane on the floor, the counts were 1790+/-2700 and 1590+/-1590 cfu/m(2)/h. For all operations the settle plates yielded 210+/-287 cfu/m(2)/h in the operating area and 720+/-564 cfu/m(2)/h in the anaesthetic area (P=0.01). Compared with the membrane placed on the anaesthetic equipment the counts on the membrane placed on patient were also significantly lower (P=0.01) while the membranes placed on the floor in each area showed no difference in counts. In conclusion, compared with conventional clothing, the use of body exhaust gowns could not be proven to provide more protection against microbial contamination. The low number of colony forming units found in the operating area was similar to that expected from an ultraclean laminar airflow unit, although achieved with a cheaper and more energy saving system.

Post-transplant lymphoproliferative syndrome in the pediatric liver transplant population.

Post-transplant lymphoproliferative disease remains a complication with a high morbidity and mortality. The present study examined 291 pediatric liver transplants performed in 263 children from October 1984 to December 1999. Post-transplant lymphoproliferative disease has an overall incidence of 12%. Tacrolimus and cyclosporine had a similar incidence of post-transplant lymphoproliferative disease. Fifty-six per cent of patients who developed post-transplant lymphoproliferative disease were Epstein-Barr virus negative at the time of transplantation. Mean time of conversion to Epstein-Barr virus positivity was 1.1 years after liver transplantation. Ten per cent of those who developed post-transplant lymphoproliferative disease never had Epstein-Barr virus detected. Mean time from Epstein-Barr virus positivity to detection of post-transplant lymphoproliferative disease was 2.68 years, and 3.13 years from liver transplantation (OLTx) to post-transplant lymphoproliferative disease. There was a 35% incidence of mortality. Deaths occurred a mean of 0.76 years after diagnosis of post-transplant lymphoproliferative disease. Most cases of post-transplant lymphoproliferative disease had extranodal location. There was one recurrence in 10% of patients, and two in 3%. All recurrent cases were seen in recipients who became Epstein-Barr virus positive after transplantation. There has been a decrease in the incidence of post-transplant lymphoproliferative disease from 15% to 9% to 4%. Post-transplant lymphoproliferative disease should be diagnosed promptly and treated aggressively. The best treatment, however, seems to be prevention, starting in the immediate postoperative period. Survivors should be monitored for both recurrence of post-transplant lymphoproliferative disease and acute cellular rejection.

[Genotypic sex and phenotypic sex: clinical, biochemical and molecular aspects in a patient with male hypogonadism and 46XX-45XO karyotype]. / Sesso genotipico e sesso fenotipico: aspetti clinici, biochimici e molecolari in un paziente con ipogonadismo maschile e cariotipo 46XX-45X0.

We report here the case of a patient with primary male ipogonadism, with small testes and deficient virilization of the external genitalia, but with 46XX, 45X0 karyotype. Hormonal determinations showed high LH and FSH and low testosterone levels. Ultrasonography confirmed the presence of small testes within the scrotum. Cytogenetic analysis demonstrated a female karyotype, with 90% 46XX, 10% 45X0 mosaicism. Using DNA probes for genes located on the Y chromosome, the presence of the "Sex-Determining Region" of the Y chromosome (SRY) was evidenced in the genomic DNA of this patient. By Fluorescence in Situ Hybridization (FISH), SRY locus was localized in the p terminal region of an X chromosome. SRY is the primary inducer of testis development; it acts as a transcription factor leading to a sequence of gene activations critical in the process of testicular differentiation and morphogenesis. A condition characterized by testicular development in subjects who lack a normal Y chromosome has been described; most of these patients are carriers of the short arm of the Y chromosome transferred to one of the two X chromosome, suggesting a form of X-Y paternal interchange. In our patient, the development of male gonade in the absence of an Y chromosome was explained by the demonstration of the SRY gene in an X chromosome.