[Novel compound heterozygous LoF mutations in SRD5A2 may result in disorders of sex development].

Objective: To investigate the novel genetic cause associated with hypospadias and the strategy for preventing offspring genetic defects in these patients. Methods: In March 2019, a patient with gonadal dysplasia (hypospadias associated with cryptorchidism) was referred to Shanghai General Hospital. His secondary sex characters, level of sex hormones and the development of male reproductive system was assessed through physical examination, sex hormone examination, male reproductive system B-ultrasound and computed tomography (CT). Whole-exome sequencing (WES) was preformed to investigate the pathogenic genetic variations associated with hypospadias and cryptorchidism. Also, Sanger sequencing was conducted to verify the WES results in the pedigree. Semen analysis was used to assess the fertility of the proband and the SRD5A2 gene analysis of his spouse was performed to assess the risk of genetic defects in the offspring. Results: The patient suffered from gonadal dysplasia (hypospadias associated with cryptorchidism). Physical examination showed an inverted triangular distribution of pubic hair, small penis and the volume of the testis was 8 ml. Sex hormone examination revealed the level of FSH, LH, Pituitary prolactin (PRL), estrogen (E(2)), testosterone (T), and sex hormone-binding globulin (SHBG) was 25.81 U/L, 10.84 U/L, 21.09 µg/L, 153 pmol/L, 16.95 nmol/L, and 36.15 nmol/L respectively. B-ultrasound and computed tomography (CT) showed left inguinal testis. Also, semen analysis illustrated that the volume was 0.05 ml and sperm concentration<2×10(6)/ml, suggesting oligospermia in this case. WES sequencing and Sanger sequencing showed compound heterozygous LoF mutations in SRD5A2 [NM_000348.3:C.679C>T(p.Arg227Ter) and NM_000348.3:C.16C>T(p.Gln6Ter)] in this patient. And there were no pathogenic genetic variations of SRD5A2 in the spouse. Conclusion: Novel compound heterozygous LoF mutations in SRD5A2[NM_000348.3:C.679C>T(p.Arg227Ter) and NM_000348.3:C.16C>T(p.Gln6Ter)] may be the primary cause of disorders of sex development.

[Efficacy and safety study of microsurgical varicocelectomy in the treatment of non-obstructive azoospermia with varicocele].

Objective: To discuss the efficacy and safety of subinguinal microsurgical varicocelectomy in the treatment of non-obstructive azoospermia (NOA) with varicocele. Methods: The clinical data of 141 patients with NOA and varicocele who underwent subinguinal microsurgical varicocelectomy from March 2015 to June 2017 in Shanghai General Hospital was collected.One hundred and ten patients suffered from varicocele on the left side, 1 on the right side, and the rest (30 cases) were bilateral varicocele. Grade Ⅰ varicocele were found on 7 sides (the right and left side was count respectively), grade Ⅱ on 121 sides, and grade Ⅲ on 43 sides. Sperm analysis, pregnancy rate and complications were recorded after at least 6 months since operation. Results: Eleven cases were lost during the follow-up. Eighteen of the remaining 130 NOA patients processed successful sperm retrieval in post-operative semen analysis (18/130, 13.8%). Six couples(6/130, 4.6%) succeeded in natural pregnancy. Five couples (5/130, 3.8%)underwent successful pregnancy following with intracytoplasmic sperm injection(ICSI). Twenty-six out of the remaining 112 patients underwent the micro dissection testicular sperm extraction (micro-TESE), and 4 patients got a successful sperm retrieval (4/26, 15.4%). Among them, 2 couples had successful pregnancy with ICSI. Totally 2 cases of postoperative infection of incision were found. Conclusions: Microsurgical varicocelectomy had a beneficial effect on sperm quality of patients suffered from NOA with varicocele to some extent, even leading to unassisted pregnancy or avoiding micro-TESE before ICSI. Microsurgical varicocelectomy could be applied in the treatment of NOA with varicocele.

The MNS glycophorin variant GP.Mur affects differential erythroid expression of Rh/RhAG transcripts.

BACKGROUND: The band 3 macrocomplex (also known as the ankyrin-associated complex) on the red cell membrane comprises two interacting subcomplexes: a band 3/glycophorin A subcomplex, and a Rh/RhAG subcomplex. Glycophorin B (GPB) is a component of the Rh/RhAG subcomplex that is also structurally associated with glycophorin A (GPA). Expression of glycophorin B-A-B hybrid GP.Mur enhances band 3 expression and is associated with lower levels of Rh-associated glycoprotein (RhAG) and Rh polypeptides. The goal of this study was to determine whether GP.Mur influenced erythroid Rh/RhAG expression at the transcript level. MATERIALS AND METHODS: GP.Mur was serologically determined in healthy participants from Taitung County, Taiwan. RNA was extracted from the reticulocyte-enriched fraction of peripheral blood, followed by reverse transcription and quantitative PCR for RhAG, RhD and RhCcEe. RESULTS: Quantification by real-time PCR revealed significantly fewer RhAG and RhCcEe transcripts in the reticulocytes from subjects with homozygous GYP*Mur. Independent from GYP.Mur, both RhAG and RhD transcript levels were threefold or higher than that of RhCcEe. Also, in GYP.Mur and the control samples alike, direct quantitative associations were observed between the transcript levels of RhAG and RhD, but not between that of RhAG and RhCcEe. CONCLUSION: Erythroid RhD and RhCcEe were differentially expressed at the transcript levels, which could be related to their different degrees of interaction or sensitivity to RhAG. Further, the reduction or absence of glycophorin B in GYP.Mur erythroid cells affected transcript expressions of RhAG and RhCcEe. Thus, GPB and GP.Mur differentially influenced Rh/RhAG expressions prior to protein translation.

[Correlation between anatomical factors of spermatic vessels and varicocele].

Objective: To analyze the correlation between anatomy of spermatic vessels and varicocele, providing reference for the preoperative assessment and treatment of varicocele. Methods: A total of 156 patients who underwent microsurgical left subinguinal varicocelectomy at Shanghai General Hospital between May 2015 and July 2016 were included in this study. The severity of varicocele and number of spermatic vessels detected in operations were recorded. According to the number of internal spermatic arteries (ISAs), the patients were divided into three groups: single-ISA group (55 cases), double-ISAs group (63 cases) and multi-ISAs group (38 cases), to analyze the correlation among spermatic vessels and to compare varicocele grade, the volume of testes, the parameter of semen analysis, serum reproductive hormone, surgery time, and hospital stay among the three groups. Results: The number of ISAs was positively correlated with the ipsilateral internal spermatic veins (ISVs) (r=0.210; P=0.008)and lymphatic vessels (r=0.224; P=0.005); the number of lymphatic vessels was positively correlated with the ipsilateral gubernacular veins (r=0.172; P=0.032)and ISVs (r=0.296; P=0.000) . The number of ISVs in the multi-ISAs group (10.58±4.28) was significantly larger than that in the single-ISA group (8.22±3.10, P=0.003). The number of lymphatic vessels in the multi-ISAs group(4.11±1.90)was also significantly larger than that in the double-ISA group(3.76±1.40, P=0.020) and the single-ISA group(3.13±1.52, P=0.007). The number of ISVs in grade 2 varicocele patients (9.74±3.90) was significantly higher than that in grade 3 varicocele patients (8.33±3.10, P=0.013). No significant differences in varicocele grade, change of pre- and post-operative semen analysis, serum reproductive hormone, the volume of ipsilateral testes, surgery time, and hospital stay were observed among the three groups. Conclusions: There is a correlation among various kinds of spermatic vessels. Patients with grade 2 varicocele, especially who have multiple ISAs, are likely to have more ISVs and lymphatic vessels. For these patients, surgeons should pay more attention to protect spermatic arteries and lymphatics carefully while ligating varicose veins completely to prevent recurrence and complications.

Dissecting alternative splicing in the formation of Miltenberger glycophorin subtype III (GYP.Mur).

BACKGROUND AND OBJECTIVES: Miltenberger subtype III (Mi.III, GP.Mur) is one of the most important red cell phenotypes in the fields of transfusion in South-East Asia. GP.Mur is believed to evolve from homologous gene recombination events between glycophorin A (GYPA) and glycophorin B (GYPB). GYP.Mur differs from GYPB in only seven nucleotides dispersed near the region of 3' exon 3 of GYP.Mur. The goal of this study was to dissect how these nucleotide variants affected splicing of exon 3. MATERIALS AND METHODS: We first designed two minigene constructs: one containing GYP.Mur from exon 2 to exon 4 and the other containing GYPB in the same region. To test how these nucleotide variations between GYP.Mur and GYPB affected the splicing, a repertoire of the GYP.Mur-like minigene constructs with different point mutations were created. These minigene variants were evaluated for their abilities to induce splicing of exon 3 using a heterologous expression system. RESULTS: (1) GYP.Mur minigene expressed exons 2, 3 and 4, whereas GYPB minigene expressed only exon 2 and exon 4. (2) The single nucleotide alteration at the position of the 5' splice site of glycophorin intron 3 reversed the splicing decision. (3) The nucleotide variations between GYP.Mur and GYPB other than that at the 5' splice site showed very little or no effect on splicing of exon 3. CONCLUSION: Splicing of the glycophorin B-A-B hybrids (GYP.Mur and GYP.BUN) and unsplicing of GYPB follow the GU-AG rule strictly.

Differential regulation of collagen, lysyl oxidase and MMP-2 in human periodontal ligament cells by low- and high-level mechanical stretching.

BACKGROUND AND OBJECTIVE: Mechanical stretching modulates extracellular matrix (ECM) protein synthesis by periodontal ligament (PDL) cells. However, the mechanoregulation of lysyl oxidase (LOX), a key enzyme for collagen cross-linking, is not fully understood. In the present study, we hypothesized that low-level and high-level mechanical stretching differentially regulates collagen deposition and the expression of LOX and the enzymes responsible for ECM degradation, such as MMP-2 in PDL cells. MATERIAL AND METHODS: Human PDL cells were cultured on flexible-bottom culture plates and subjected to cyclic mechanical stretching (3% and 10% elongation at 0.1 Hz) for 24 and 48 h in a Flexercell FX-4000 strain unit. The levels of expression of type I collagen alpha 1 (COL1A1), type III collagen alpha 1 (COL3A1), lysyl oxidase (LOX), MMP2 and TIMP2 mRNAs were analyzed using an RT-PCR technique. The cell layer and the culture medium were separately collected and processed for detection of the following ECM-related molecules: (i) total collagen content using a Sircol dye-binding method; (ii) LOX protein expression by western blotting; (iii) LOX activity using a fluorometric assay; and (iv) MMP-2 enzyme activity by gelatin zymography. RESULTS: Low-level (3%) mechanical stretching of PDL cells upregulated the expression of COL1A1, COL3A1 and LOX mRNAs, enhanced the production of collagen and increased the LOX activity but did not change the level of expression of MMP2 or TIMP2 mRNA. The collagen content and LOX activity showed obvious elevation in the medium, but not in the cell layer. High-level (10%) mechanical stretching downregulated COL1A1 mRNA but upregulated COL3A1 mRNA; however, the effect on COL3A1 was smaller, and occurred earlier, compared with the effect on the COL1A1 gene. High-level mechanical stretching upregulated the expression of MMP2 and TIMP2 mRNAs but did not change collagen production or LOX activity. Moreover, high-level mechanical stretching increased the level of pro-MMP-2, especially in the cell layer. CONCLUSIONS: This study substantiates the mechanoregulation of the expression of ECM-related molecules in PDL cells. High-level mechanical stretching upregulated the expression of MMP2 and TIMP2 mRNAs, but did not affect collagen production or LOX activity. In addition to increasing the transcription of COL1A1, COL3A1 and LOX genes, low-level mechanical stretching enhanced total collagen production and LOX activity, which should favor ECM stabilization. As an effective regulator of ECM remodeling, mechanical stretching can be exploited in periodontal regeneration and ligament tissue engineering via application of appropriate mechanical stimulation.

Characterization of facial asymmetry in skeletal Class III malocclusion and its implications for treatment.

Jaw deviation is frequently seen in Class III patients. The aim of the study was to investigate asymmetric features of skeletal, dental and soft tissues in three types of jaw asymmetry based on our previously reported classification system. The cone-beam computed tomography (CBCT) images of 70 Class III patients were analysed. Group 1 patients showed large shift of menton and synchronous but smaller ramus deviation. The maxillomandibular complex had roll and yaw rotations to the menton-deviation side. Maxillary and dental asymmetry was obvious in transverse and vertical dimensions. Cant of occlusal plane and lip line was apparent. Group 2 patients also exhibited menton and ramus deviation to the same side but the discrepancy in ramus width was larger than menton shift. Asymmetry in Group 2 resulted from a bodily side shift of the maxillomandibular complex without obvious rotation. Group 3 patients had menton and ramus deviated in opposite directions which seemed secondary to a yaw rotation. Double-jaw surgery is generally required for Groups 1 and 3 while Group 2 patients may be successfully treated by mandibular surgery only provided that arch width discrepancy can be managed by orthodontic measures.

MMP-3 response to compressive forces in vitro and in vivo.

During orthodontic tooth movement, bone resorption occurs at the compression site. However, the mechanism underlying resorption remains unclear. Applying compressive force to human osteoblast-like cells grown in a 3D collagen gel, we examined gene induction by using microarray and RT-PCR analysis. Among 43 genes exhibiting significant changes, cyclo-oxygenase-2, ornithine decarboxylase, and matrix metalloproteinase-3 (MMP-3) were up-regulated, whereas membrane-bound interleukin-1 receptor accessory protein was down-regulated. The MMP-3 protein increases were further confirmed by Western blot. To ascertain whether MMP-3 is up-regulated in vivo by orthodontic force, we examined human bone samples at the compressive site by realigning the angulated molars. Immunohistochemical staining revealed MMP-3 distributed along the compressive site of the bony region within 3 days of compression. Since MMP-3 participates in degradation of a wide range of extracellular matrix molecules, we propose that MMP-3 plays an important role in bone resorption during orthodontic tooth movement.

A Salmonella infection complicated with suppurative thyroiditis and ruptured aortic mycotic aneurysm in a renal transplant recipient.

We report a renal transplant recipient who presented with fever and chills for 2 days. The blood and stool cultures revealed the growth of Salmonella enteriditis. A whole-body gallium scan played an important role in the subsequent diagnosis of suppurative thyroiditis. To our knowledge, this is the first report of acute S. enteriditis thyroiditis in a renal transplant recipient. Despite vigorous antibiotic use and a partial thyroidectomy, he experienced recurrent S. enteriditis infection, resulting in a ruptured thoracic mycotic aneurysm 1 month later. Finally the patient was successfully cured with aneurysm resection, in situ reconstruction of the thoracic aorta, and prolonged antibiotics.

Abdominal wall abscess secondary to spilled gallstones: late complication of laparoscopic cholecystectomy and preventive measures.

Spilled gallstones left in the abdominal cavity or trapped at trocar sites may cause considerable morbidity. We saw a patient with an abdominal wall abscess 2 years after laparoscopic cholecystectomy secondary to spilled stones. After we reviewed the operative procedure in addition to the accumulated experience in laparoscopic surgery, we believe that retrieval of specimens and their contents is of paramount importance, especially when the gallbladder is infected, contains several stones, or may harbor malignancy. Therefore, we made use of a simple surgical glove with a long pursestring suture surrounding the opening to collect the specimen. This method proved to be simple and quite convenient, with the needed materials readily available. It can collect the spilled stones within the abdominal cavity as well as the gallbladder and can transport these stones out of the abdominal cavity with ease and safety. It also protects the specimen in contact with the wound and cuts short the operating time. The technique and advantages are described.

Laparoscopic appendectomy for ruptured appendicitis.

We conducted a retrospective analysis to assess the feasibility of laparoscopic appendectomy in cases of ruptured appendicitis. Between August 1993 and April 1998, a total of 328 laparoscopic appendectomies were performed in Min-Shen General Hospital. There were 34 cases of pathology-proven ruptured appendicitis. Patients were divided into three groups according to the operative findings: group 1 (10 cases) consisted of patients with a perforated appendix with local peritonitis, group 2 (15 cases) consisted of patients with perforated appendix with diffused peritonitis, and group 3 (9 cases) consisted of patients with abscess formation around the perforated appendix. Three cases in group 3 were converted to laparotomy and were excluded from this study. Mean age (+/- SD) was 30 +/- 15 years in group 1, 39 +/- 23 years in group 2, and 37 +/- 13 years in group 3. Duration of symptoms was longer in group 3 (4.2 +/-1.2 days) than in group 1 (1.8 +/- 1.3 days) and group 2 (2.3 +/- 1.2 days). There was no difference in operation time among groups 1 (52 +/- 10 min), 2 (64 +/- 13 min), and 3 (67 +/- 16 min). The time of flatus passage after operation was similar in the three groups (group 1, 17 +/- 11 hours; group 2, 21 +/- 12 hours; group 3, 24 +/- 8 hours). Hospital stay was significantly shorter in group 1 (3.0 +/- 1.1 days) than in group 2 (5.1 +/- 2.2 days) and group 3 (4.2 +/- 1.2 days). There were no complications and no readmissions. Our results indicate that the laparoscopic approach is feasible for ruptured appendicitis with local or diffuse peritonitis and in selected cases with abscess formation. However, prospective randomized controlled trials are needed to determine which procedure is to be recommended.

Liberal use of a bag made from a surgical glove during laparoscopic surgery for specimens retrieval.

Laparoscopic surgery has been gaining wide acceptance in recent years. Tissue removal from the abdominal cavity becomes a paramount issue, especially when the specimen is infected, contains several fragments, or potentially contains a malignancy. Several sophisticated instruments and devices have been designed for this purpose. However, most of these are expensive, difficult to handle, and come in a fixed size. Condoms, glove fingers, and zipper-type plastic bags have been tried also, but with limited success. The authors report use of a simple bag made from a surgical glove to collect specimens; a long purse-string suture surrounds the opening or the base of the glove finger to secure material during retrieval. This method has been found to be simple and convenient, and necessary construction material is readily available. The bag not only protects the specimen from contact with the wound but also cuts short the operating time. The technique and advantages are described.

Small bowel perforation caused by a sharp bone: laparoscopic diagnosis and treatment.

Small bowel perforation caused by sharp or pointed foreign bodies rarely is diagnosed preoperatively because clinical symptoms usually are nonspecific and can mimic other surgical conditions such as appendicitis and cecal diverticulitis. We report the case of a 57-year-old man who underwent diagnostic laparoscopy for abdominal pain of unknown etiology. Swelling and erythema were noted in a segment of small bowel in the right lower abdomen. A tiny sharp-pointed object was found penetrating the inflamed portion of the bowel. This foreign body was removed and the perforation immediately repaired under laparoscopic guidance. The postoperative course was uneventful. We believe that in cases of lower abdominal pain with peritoneal irritation, laparoscopy can provide not only a wide field of examination to localize intraabdominal pathology, but in some instances it can aid in case management.

Laparosopic removal of large gastric phytobezoars.

Large gastric bezoars are difficult to remove endoscopically. A 78-year-old man presenting with abdominal pain and loss of appetite for 4 months was admitted and evaluated. Gastroscopy disclosed two large phytobezoars within the stomach. Laparoscopic removal was undertaken. The bezoars were removed via a gastrotomy using the three-trocar technique. They were successfully retrieved from the abdominal cavity using an improvised "endobag" made from a simple surgical glove. Such an endobag presents several advantages; they are easy to make, sterile, economical, readily available, disposable, there is ample space to manipulate the specimen within, and there is minimal risk of contamination throughout the procedure. The authors recommend this approach for the treatment of patients with large gastric bezoars in whom laparotomy is indicated.

Laparoscopic resection of an omental cyst with pedicle torsion.

Omental cysts are the least-common variety of all types of intra-abdominal cystic lesions. In the past, transabdominal laparotomy with excision of the cyst was the treatment of choice. With the advent of laparoscopic surgery, it has become possible to resect the cyst without the need for a large incision in the abdomen. We report a case of a 15-year-old girl who underwent diagnostic laparoscopy for recurring abdominal pain of 2 years' duration. The procedure revealed a huge cyst lying above the omentum with its pedicle rising from the greater curvature of the stomach. The pedicle was noted to have twisted eight times in a clockwise direction. The cyst was resected by laparoscopic means using three trocars. The postoperative course was uneventful. As presented in this case, we believe that a laparoscopic approach is an attractive alternative for the management of omental cystic lesions.

Subpopulations of intrathyroidal lymphocytes in Graves' disease.

Subsets of T- and B-lymphocytes were immunophenotyped in frozen thyroid tissues from 20 patients with Graves' disease by using the avidin-biotin-complex peroxidase method. A panel of B- and T-cell monoclonal antibodies were employed to detect the subsets of T cells, activated T cells, natural killer cells and B cells. Serum thyroglobulin and microsomal antibodies were also measured simultaneously from both the thyroid vein and the peripheral vein in 9 patients. The quantitative results found that an almost equal ratio of lymphocytes stained positively for Leu 3a (CD4) and T8 (CD8) in almost all specimens. Activated lymphocytes expressing T9, T10, and Tac (CD25) were sporadically noted in the interstitial areas. The lymphoid aggregates and follicles were B cell clusters. Natural killer cells appear to play an insignificant role in Graves' disease. The thyroid epithelial cells expressed HLA-DR (Ia+) and HLA-ABC, suggesting a possible localized immune reaction between antigen-presenting thyroid epithelial cells and lymphocytes. The titers of serum thyroglobulin and microsomal antibodies from either the thyroid vein or the peripheral vein showed no definite correlation with the lymphoid distribution of the thyroid tissues, implying that the in situ or local immunologic reaction may not reflect the status of systemic antibody production. The immunologic mechanism and pathogenesis of Graves' disease is reviewed and discussed.

Rhodanese and sodium thiosulfate encapsulated in mouse carrier erythrocytes. II. In vivo survivability and alterations in physiologic and morphologic characteristics.

Biodegradable drug carrier mechanisms were employed in drug antagonism studies. Prior studies indicated that erythrocytes containing encapsulated rhodanese and sodium thiosulfate metabolized cyanide to thiocyanate in vitro. Studies were conducted to investigate the properties of these sulfurtransferase-loaded red blood cells in vivo by administering the carrier red blood cells intravenously. Approximately 40 to 50% of the cells were eliminated within the first few hours while the remaining loaded erythrocytes persisted in the circulation. The present studies were initiated to investigate the characteristics of the disposition of the loaded erythrocytes and to examine differences in the properties between carrier and noncarrier erythrocytes. Also, the disposition and viability of the erythrocytes in vivo were studied with relation to various biochemical, physiological, and morphological properties. These studies indicated that the carrier erythrocytes had a smaller cell volume and were more susceptible to hemolysis than normal erythrocytes. Morphologic studies by electron microscopy indicated that extensive morphologic changes occurred during the procedures after hypotonic dialysis, isotonicity adjustment, and resealing were completed. Differences were noted between those cells that were only resealed and those cells that were also subjected to annealing. The morphologic characteristics of most of the cells were restored to the "normal" morphologic appearance only after annealing. Annealed erythrocytes' in vivo survivability was correlated with the physical properties of these cells.