Genomic testing for copy number and single nucleotide variants in spermatogenic failure.

PURPOSE: To identify clinically significant genomic copy number (CNV) and single nucleotide variants (SNV) in males with unexplained spermatogenic failure (SPGF). MATERIALS AND METHODS: Peripheral blood DNA from 97/102 study participants diagnosed with oligozoospermia, severe oligozoospermia, or non-obstructive azoospermia (NOA) was analyzed for CNVs via array comparative genomic hybridization (aCGH) and SNVs using whole-exome sequencing (WES). RESULTS: Of the 2544 CNVs identified in individuals with SPGF, > 90% were small, ranging from 0.6 to 75 kb. Thirty, clinically relevant genomic aberrations, were detected in 28 patients (~ 29%). These included likely diagnostic CNVs in 3/41 NOA patients (~ 7%): 1 hemizygous, intragenic TEX11 deletion, 1 hemizygous DDX53 full gene deletion, and 1 homozygous, intragenic STK11 deletion. High-level mosaicism for X chromosome disomy (~ 10% 46,XY and ~ 90% 47,XXY) was also identified in 3 of 41 NOA patients who previously tested normal with conventional karyotyping. The remaining 24 CNVs detected were heterozygous, autosomal recessive carrier variants. Follow-up WES analysis confirmed 8 of 27 (30%) CNVs (X chromosome disomy excluded). WES analysis additionally identified 13 significant SNVs and/or indels in 9 patients (~ 9%) including X-linked AR, KAL1, and NR0B1 variants. CONCLUSION: Using a combined genome-wide aCGH/WES approach, we identified pathogenic and likely pathogenic SNVs and CNVs in 15 patients (15%) with unexplained SPGF. This value equals the detection rate of conventional testing for aneuploidies and is considerably higher than the prevalence of Y chromosome microdeletions. Our results underscore the importance of comprehensive genomic analysis in emerging diagnostic testing of complex conditions like male infertility.

Testicular tissue cryopreservation: 8 years of experience from a coordinated network of academic centers.

STUDY QUESTION: Is it feasible to disseminate testicular tissue cryopreservation with a standardized protocol through a coordinated network of centers and provide centralized processing/freezing for centers that do not have those capabilities? SUMMARY ANSWER: Centralized processing and freezing of testicular tissue from multiple sites is feasible and accelerates recruitment, providing the statistical power to make inferences that may inform fertility preservation practice. WHAT IS KNOWN ALREADY: Several centers in the USA and abroad are preserving testicular biopsies for patients who cannot preserve sperm in anticipation that cell- or tissue-based therapies can be used in the future to generate sperm and offspring. STUDY DESIGN, SIZE, DURATION: Testicular tissue samples from 189 patients were cryopreserved between January 2011 and November 2018. Medical diagnosis, previous chemotherapy exposure, tissue weight, and presence of germ cells were recorded. PARTICIPANTS/MATERIALS, SETTING, METHODS: Human testicular tissue samples were obtained from patients undergoing treatments likely to cause infertility. Twenty five percent of the patient's tissue was donated to research and 75% was stored for patient's future use. The tissue was weighed, and research tissue was fixed for histological analysis with Periodic acid-Schiff hematoxylin staining and/or immunofluorescence staining for DEAD-box helicase 4, and/or undifferentiated embryonic cell transcription factor 1. MAIN RESULTS AND THE ROLE OF CHANCE: The average age of fertility preservation patients was 7.9 (SD = 5) years and ranged from 5 months to 34 years. The average amount of tissue collected was 411.3 (SD = 837.3) mg and ranged from 14.4 mg-6880.2 mg. Malignancies (n = 118) were the most common indication for testicular tissue freezing, followed by blood disorders (n = 45) and other conditions (n = 26). Thirty nine percent (n = 74) of patients had initiated their chemotherapy prior to undergoing testicular biopsy. Of the 189 patients recruited to date, 137 have been analyzed for the presence of germ cells and germ cells were confirmed in 132. LIMITATIONS, REASONS FOR CAUTION: This is a descriptive study of testicular tissues obtained from patients who were at risk of infertility. The function of spermatogonia in those biopsies could not be tested by transplantation due limited sample size. WIDER IMPLICATIONS OF THE FINDINGS: Patients and/or guardians are willing to pursue an experimental fertility preservation procedure when no alternatives are available. Our coordinated network of centers found that many patients request fertility preservation after initiating gonadotoxic therapies. This study demonstrates that undifferentiated stem and progenitor spermatogonia may be recovered from the testicular tissues of patients who are in the early stages of their treatment and have not yet received an ablative dose of therapy. The function of those spermatogonia was not tested. STUDY FUNDING/COMPETING INTEREST(S): Support for the research was from the Eunice Kennedy Shriver National Institute for Child Health and Human Development grants HD061289 and HD092084, the Scaife Foundation, the Richard King Mellon Foundation, the Departments of Ob/Gyn & Reproductive Sciences and Urology of the University of Pittsburgh Medical Center, United States-Israel Binational Science Foundation (BSF), and the Kahn Foundation. The authors declare that they do not have competing financial interests.

Is follow-up CT imaging of the chest and abdomen necessary after preoperative neoadjuvant therapy in rectal cancer patients without evidence of metastatic disease at diagnosis?

AIM: Patients with rectal cancer often undergo multiple CT scans prior to surgical resection. We propose that in patients with locally advanced rectal cancer without evidence of metastatic disease at presentation, CT imaging of the chest and abdomen after preoperative neoadjuvant therapy does not change clinical information or surgical management. METHOD: An institutional review board-approved medical record review identified patients with contrast enhanced CT of the chest, abdomen and pelvis alone or in conjunction with (18)F-fluoro-2-deoxy-d-glucose/positron emission tomography imaging for staging of rectal cancer prior to and after neoadjuvant therapy. Eighty-eight patients were included in the study. Scans were reviewed for the presence of metastatic disease on initial and follow-up imaging prior to surgical resection. RESULTS: Seventy-six (86%) of 88 patients had no evidence of metastasis at presentation. None of these patients developed metastatic disease after neoadjuvant therapy. Twelve (14%) had metastases at presentation. No study patient developed metastatic disease in a new organ. CONCLUSION: Imaging after preoperative neoadjuvant therapy in rectal cancer does not change the designation of metastatic disease. Patients with locally advanced rectal adenocarcinoma without evidence of metastases may not benefit from repeat imaging of the chest and abdomen after neoadjuvant therapy.

Protective ocular mechanisms in woodpeckers.

AIMS: Woodpeckers possess mechanisms protecting the eye from shaking/impact. Mechanisms available to woodpeckers but not humans may help explain some eye injuries in Shaken Baby syndrome (SBS). METHODS: Gross dissection and histologic examination of eyes and orbits of seven woodpeckers. RESULTS: All birds showed restricted axial globe movement due to the tight fit within the orbit and fascial connections between the orbital rim and sclera. The sclera was reinforced with cartilage and bone, the optic nerve lacked redundancy, and the vitreous lacked attachments to the posterior pole retina. CONCLUSIONS: Woodpecker eyes differ from human infants by an inability of the globe to move axially in the orbit, the sclera to deform, and the vitreous to shear the retina. These findings support current hypotheses that abusive acceleration-deceleration-induced ocular injury in human infants may be related to translation of vitreous within the globe and the globe within the orbit. The woodpecker presents a natural model resistant to mechanical forces that have some similarity to SBS.

[Therapeutic penetrating keratoplasty for microbial keratitis].

BACKGROUND: Microbial keratitis is a potentially sight threatening disease. Most cases respond well to antimicrobial therapy. However, in cases that progress despite intensive medical therapy, an urgent therapeutic penetrating keratoplasty (TPKP) is required. AIM: To evaluate the indications and results of TPKP in Israel. METHODS: A retrospective study reviewed the TPKP performed at the Goldschleger Eye Institute, Sheba Medical Center, between 1990-2003. The study included 18 cases of at least one-year follow-up. RESULTS: The indications for TPKP included severe infectious keratitis unresponsive to medical treatment in 33% of the patients and severe corneal destruction in 66% of them. The infectious keratitis was diagnosed as bacterial keratitis in 44% of the patients, unidentified pathogen in 39%, mycotic in 11% and acanthamoeba in 6% of the patients. Risk factors in the patients with microbial keratitis requiring TPKP included: previous ocular disease in 39%, previous ocular surgery in 66%, systemic disorders in 28% and ocular risk factors in 28% patients. TPKP was successful in bacterial and acanthamoeba keratitis as far as the transparency of the graft and elimination of the infection and improvement of visual acuity. However, TPKP failed in mycotic and unidentified keratitis. The risk factors for failure included: previous ocular disease or surgeries, systemic disorders or large corneal grafts. CONCLUSIONS: Therapeutic penetrating keratoplasty is an important and effective therapeutic tool for intractable bacterial and acanthamoeba keratitis. Prognostic factors for graft success include lack of ocular disease or previous surgeries, lack of systemic disorders or small corneal graft size.

3.0- Versus 1.5-T MR cholangiography: a pilot study.

OBJECTIVE: The purpose of our study was to evaluate quantitative and qualitative image quality of MR cholangiography at a field strength of 3.0 T compared with the standard field strength of 1.5 T. MATERIALS AND METHODS: A standardized MR cholangiography sequence protocol was used for 15 healthy male volunteers (mean age +/- SD, 32.4 +/- 4.3 years) who underwent both 1.5- and 3.0-T MRI within 2 hr in an alternating fashion. Dedicated circular polarized torso coils (1.5 and 3.0 T) were used. The sequence protocol included breath-hold single-slice rapid acquisition with relaxation enhancement (slice thickness, 50 mm; orientation, coronal and +/- 20 degrees oblique coronal); breath-hold multislice HASTE (slice thickness, 3 mm; coronal only); and a non-breath-hold, respiratory-triggered 3D turbo spin-echo (TSE) T2-weighted sequence (slice thickness, 1 mm; 60 slices per slab; coronal only). Maximum intensity projections were generated from each multislice data set. Bile duct (common bile duct, right posterior segmental branch, and left hepatic duct) to periductal tissue contrast-to-noise ratios were compared at 1.5 and 3.0 T. Qualitative image analysis was performed by three independent reviewers. Qualitative analysis included delineation of the extra- and intrahepatic biliary anatomy, with specific attention given to the presence (or absence) of cystic or intrahepatic ductal variants, using a 4-point confidence scale. Statistical analysis consisted of the paired Student's t test and the signed rank test. RESULTS: Contrast-to-noise ratios between the bile duct and the periductal tissue were higher at 3.0 T in all three locations (common bile duct, right posterior segmental branch, and left hepatic duct). In each magnet class, the 3D TSE sequence offered the best contrast-to-noise ratio and qualitative analysis. Superiority of the 3D TSE sequence was statistically significant in all analyses. Five of the 15 volunteers had intrahepatic biliary variants that were detected with a higher level of confidence (p < 0.01) on the 3.0-T system than on the 1.5-T system. CONCLUSION: Compared with MR cholangiography at 1.5 T, MR cholangiography at 3.0 T offers improved contrast-to-noise ratio and a higher level of confidence for depicting intrahepatic variants.

Endoscopic ultrasound and computer tomography are inaccurate methods of classifying cystic pancreatic lesions.

BACKGROUND: Despite advances in imaging modalities, preoperative diagnosis of pancreatic cystic lesions remains difficult. AIM: To assess the accuracy of endoscopic ultrasound and computer tomography to preoperatively distinguish benign from potentially malignant and malignant pancreatic cystic lesions. METHODS: Photograph series obtained from endoscopic ultrasound examinations of 66 patients with cystic pancreatic lesions were blindly reviewed by three endoscopic ultrasonographers. Forty-one of those 66 patients also underwent a computer tomography scan at our institution, which was blindly reviewed by a single radiologist. Computer tomography and endoscopic ultrasound classification into benign and malignant and potentially malignant pancreatic cystic lesions was correlated with the final diagnosis, which was established by surgical pathology (n = 43), diagnostic fine needle aspiration (n = 13) or follow-up imaging (n = 10). Interobserver agreement was measured using kappa statistics. RESULTS: Endoscopic ultrasound classification by the three examiners into benign versus malignant or potentially malignant cystic lesions was correct in 65-67%. Interobserver agreement was 50%. Kappa values for pairs of endoscopic ultrasound examiners were 0.16, 0.43 and 0.53. Computer tomography classification was correct in 71% and in agreement with the endoscopic ultrasound classification in 56-61% (kappa 0.12 to 0.27). CONCLUSIONS: Endoscopic ultrasound and computer tomography cannot accurately distinguish between benign pancreatic cystic lesions and malignant or potentially malignant ones. There is poor-to-modest interobserver agreement in classifying these lesions.

Percutaneous biopsy of periarterial soft tissue cuffs in the diagnosis of pancreatic carcinoma.

Pancreatic cancer may present on computed tomography (CT) as an isolated cuff of tumor surrounding the superior mesenteric artery (SMA) or celiac trunk, without an identifiable pancreatic mass. We reviewed our experience with imaging-guided biopsy of the soft tissue cuff in this patient group. A retrospective review of our interventional database identified 163 patients referred for biopsy of suspected pancreatic carcinoma. Of these, eight patients underwent biopsy of an isolated cuff of soft tissue encasing the SMA (n = 6) or celiac trunk (n = 2). None of these eight patients had an identifiable pancreatic mass. The mean width of tissue cuff biopsied was 1.3 cm (range, 0.9-2.0 cm). Nine imaging-guided biopsies were performed in eight patients. Five biopsies were performed with color Doppler ultrasound and four with CT fluoroscopy. There was a median of two needle passes per procedure (range, 1-4). In six cases, a diagnosis of pancreatic adenocarcinoma was made at the first biopsy session. In one patient, ultrasound-guided biopsy was negative, but subsequent CT-guided biopsy was positive. In one additional patient with chronic pancreatitis, biopsy revealed benign fibrous tissue. There were no procedure-related complications. In patients with suspected pancreatic cancer (but without a focal parenchymal mass), imaging-guided biopsy of isolated periarterial tissue cuffs of tumor is accurate and safe.

Evaluation of superior rectus muscle attachment following hang-back recession in rabbit eyes.

PURPOSE: The hang-back suture technique has been advocated to decrease the risk of scleral perforation during strabismus surgery. In this study, we evaluated the muscle insertion site in rabbits 4 weeks after a hang-back recession was performed. METHODS: Twenty-five eyes of 13 rabbits underwent hang-back recessions of between 5 and 8 mm of the superior rectus muscle. Four weeks later, the attachment sites were examined. RESULTS: Four weeks following the surgery, 14 muscles (n=25, 56%) were anteriorly displaced. In the 5 mm recession group, one muscle was anteriorly displaced (n=7, 14.3%). In the 6 mm recession group, 50% of the muscles recessed were displaced anteriorly (n=8). The 7 mm hang-back recession group demonstrated forward displacement in five muscles operated (n=6, 83%) and in the 8 mm recession group, all four muscles (100%) were anteriorly displaced. The magnitude of displacement was positively correlated to the amount of the hang-back recession performed (Pearson correlation p<0.001). CONCLUSIONS: Anterior displacement of muscles using the hang-back technique in the rabbit eye is unacceptably high and occurred in 56% of the cases. A positive, statistically significant correlation exists between the amount of recession performed and the number of muscles displaced from their new insertion. Therefore, when performing large recessions using the hang-back technique displacement should be anticipated.

Sclerotomy using needle puncture and subconjunctival injection of mitomycin C.

OBJECTIVE: To explore the possibilty of creating a temporary sclerotomy using a 26-gauge needle and sustaining its patency by injectiong 0.1 mL of 0.1 mg/mL of mitomycin C (MMC). ANIMALS AND METHODS: Twenty-four New Zealand white rabbits were randomized into 3 groups. In the test group, puncture was performed using 26-gauge needle penetrating the anterior chamber at the limbus, and 0.1 mg/mL MMC were injected subconjunctivally. In the control group, a puncture was performed in the same fashion and in the MMC group, MMC only was injected subconjunctivally. Intraocular pressure (IOP) was measured prior to surgery and at days 1, 3, 7, 10, 14, 21, and 28. RESULTS: The mean intraocular pressure in eyes that underwent puncture with application of MMC was significantly lower than the IOP in the eye at all measurements up to and including day 21. In the puncture group, pressure was significantly lower than in the fellow eye only at day 3. No significant pressure reduction was observed at any measurement interval in eyes treated with MMC only. CONCLUSION: This simple technique of subconjunctival puncture of the limbus combined with application of MMC was effective in lowering IOP in rabbits. This may serve to attain a temporary filter in patients who need strict short-term pressure control.

The reculture technique: individualizing the treatment of Acanthamoeba keratitis.

PURPOSE: To assess the efficacy of three drugs in different concentrations against different strains of Acanthamoeba using the reculture technique. METHODS: Cysts and trophozoites were immersed in five separate solutions. The solutions administered included 0.1% and 0.02% polyhexamethylene biguanide (PHMB), 0.1% and 0.02% chlorhexidine, and propamidine isethionate (Brolene). Readings took place after 1, 5, and 24 hours. The cysts and trophozoites were then recultured for an additional period of 48 hours. An effective drug was defined as a medication that inhibited any growth of trophozoites using the reculture technique. RESULTS: Chlorhexidine at concentrations of 0.1% and 0.02% was the only effective drug against all five strains of Acanthamoeba examined, and no trophozoites were detected on plates immersed with this agent. Only 0.1% chlorhexidine was effective in destroying all cysts in the five strains examined. CONCLUSION: We found that 0.02% chlorhexidine was efficient in irradicating all trophozoites and 0.1% chlorhexidine was effective in eradicating all cysts in the samples we examined. Therefore, it may be possible that 0.02% chlorhexidine is a good initial treatment in amoebic keratitis. Sensitivity testing, then, may be performed using the reculture technique and specification of therapy can be made accordingly.

Strabismus surgery using the adjustable suture technique.

BACKGROUND AND OBJECTIVE: To determine the results of adjustable suture technique used in horizontal muscle surgery. MATERIALS AND METHODS: Seventy-eight charts of patients who underwent strabismus surgery between the years 1993 and 1995 were examined retrospectively. The study included 35 cases of esotropia (ET), and 43 cases of exotropia (XT). The results of strabismus surgery were measured and compared 1 day after adjustment; the final results between 6 and 24 months after the surgical procedure. RESULTS: Adjustment was required in 39% of all patients. The highest rate of adjustment was required in patients who underwent monocular surgery for XT (51%), and the lowest rate of adjustment was undertaken in patients who underwent monocular surgery for ET (16%). Mean changes in the angle of deviation between 1 day after surgery to the last follow up was 2.2 prism diopters (delta) (+/-11.2 delta) for cases of esotropia, and 4.6 delta (+/-8.7 delta) for exotropia. The most significant drift was found in patients with XT who underwent binocular surgery 6.8 delta (+/-0.9 delta) and the smallest drift was found in patients with ET who underwent binocular surgery 5.2 delta (+/-5.6 delta). CONCLUSION: Esotropic and exotropic patients have a tendency to drift towards their original deviation postoperatively. It is possible, therefore, that mild overcorrection in the early postoperative period will result in better long-term results.

Sperm pellet analysis: a technique to detect the presence of sperm in men considered to have azoospermia by routine semen analysis.

PURPOSE: In men considered to have azoospermia by routine semen analyses sperm may be identified after centrifuging the semen. Because these sperm may be used for intracytoplasmic sperm injection, we describe our technique and findings of sperm pelleting. MATERIALS AND METHODS: Semen centrifugation for sperm pellet analysis was performed in 140 consecutive men in whom no sperm was identified on routine semen analysis and who were categorized as having obstructive or nonobstructive azoospermia. Obstructive azoospermia was defined as failed vasectomy reversal, failed reconstruction for congenital vasal or epididymal occlusion, or an acquired obstruction unrelated to ejaculatory duct obstruction. Patients with congenital absence of the vas deferens or who had undergone vasectomy were not included in the study. Nonobstructive azoospermia was defined as moderate to severe testicular atrophy with markedly elevated serum follicle-stimulating hormone (greater than 3 times normal), or a testicular biopsy that revealed maturational arrest, severe hypospermatogenesis or the Sertoli-cell-only pattern. Obstructive and nonobstructive azoospermia were present in 70 men who provided 109 samples and 70 who provided 103, respectively. RESULTS: Motile and nonmotile sperm was identified in 13 of the 70 patients (18.6%) with obstructive and in 16 of the 70 (22.8%) with nonobstructive azoospermia. Pellet variability, that is the absence of sperm in 1 specimen and its presence in another from the same patient, was noted in 7 of the 17 men (41.2%) with obstructive and 2 of the 17 (11.8%) with nonobstructive azoospermia (not statistically significant). Motile sperm was present in the pellets of 6 of the 70 men (8.6%) with obstructive and 15 of the 70 (21.4%) with nonobstructive azoospermia. The median number of motile sperm was lower in the obstructive than in the nonobstructive group (0 sperm in 17 samples versus 5 sperm in 41 samples, p <0.001). The median value of 0 in the obstructive azoospermia group reflects the finding that 9 of the 17 samples did not contain motile sperm. Similarly the median number of nonmotile sperm was lower in the obstructive than in the nonobstructive group (5 versus 8 sperm). CONCLUSIONS: We demonstrated the presence of motile and nonmotile sperm in a significant number of men considered to have azoospermia by routine semen analysis. Semen centrifugation (sperm pelleting) should be performed in all men considered to have this condition by routine semen analysis, especially those with testicular failure and those in whom intracytoplasmic sperm injection is possible.

Intraocular pressure increments after cataract extraction in glaucomatous eyes with functioning filtering blebs.

BACKGROUND AND OBJECTIVE: The effects of cataract extraction on intraocular pressure (IOP) were studied in glaucoma patients who had previously undergone trabeculectomy. PATIENTS AND METHODS: The files of 22 patients with 25 glaucomatous eyes with functioning filtering blebs who underwent cataract extraction were retrospectively reviewed. All patients had undergone pretrabeculectomy examination and had at least 18 months of follow-up after cataract surgery. Visual acuity, IOP, status of the filtering bleb, and the number of medications applied were recorded. RESULTS: The mean increase in IOP was 3.63 mm Hg, 3.84 mm Hg, 5.4 mm Hg, and 2.8 mm Hg at 3, 6, 12, and 18 months after cataract extraction, respectively. This elevation was statistically significant 3 months postoperatively (P < .001) and remained relatively constant thereafter. The postoperative IOP was still significantly lower than the pretrabeculectomy IOP (P < .001). CONCLUSIONS: Cataract extraction through corneal incisions in patients with functioning filtering blebs is followed by an increase in IOP. Cataract surgery in these patients does not neutralize the pressure-lowering effect achieved by the trabeculectomy, but it tends to elevate the post-trabeculectomy baseline pressure.

Diagnosis of postoperative urinary ascites using renal scintigraphy.

Nuclear medicine biliary studies have been routinely used to identify bile leaks that occur after laparoscopic cholecystecomy. The use of a Tc-99m mercaptoacetyltriglycine (MAG3) renal scan to diagnose a case of urinary leakage that occurred after a laparoscopic-assisted colectomy is shown in this report. Laparoscopic surgery is widely used in place of conventional laparotomy to minimize recovery time and discomfort after surgery. The complication rate for laparoscopic colectomy has been reported as approximately 6% to 10%. In particular, ureteral leak has been reported in 2% of procedures. Ascites of unknown origin can become a diagnostic dilemma. We present a case of postoperative ascites of unknown origin that was successfully diagnosed as urinary leakage using renal scintigraphy.

Mouse autosomal homolog of DAZ, a candidate male sterility gene in humans, is expressed in male germ cells before and after puberty.

Deletion of the Azoospermia Factor (AZF) region of the human Y chromosome results in spermatogenic failure. While the identity of the critical missing gene has yet to be established, a strong candidate is the putative RNA-binding protein DAZ (Deleted in Azoospermia). Here we describe the mouse homolog of DAZ. Unlike human DAZ, which is Y-linked, in mouse the Dazh (DAZ homolog) gene maps to chromosome 17. Nonetheless, the predicted amino acid sequences of the gene products are quite similar, especially in their RNP/RRM (putative RNA-binding) domains, and both genes are transcribed predominantly in testes; the mouse gene is transcribed at a lower level in ovaries. Dazh transcripts were not detected in testes of mice that lack germ cells. In testes of wildtype mice, Dazh transcription is detectable 1 day after birth (when the only germ cells are prospermatogonia), increases steadily as spermatogonial stem cells appear, plateaus as the first wave of spermatogenic cells enters meiosis (10 days after birth), and is sustained at this level thereafter. This unique pattern of expression suggests that Dazh participates in differentiation, proliferation, or maintenance of germ cell founder populations before, during, and after the pubertal onset of spermatogenesis. Such functions could readily account for the diverse spermatogenic defects observed in human males with AZF deletions.