Assessment of the need for follicle stimulating hormone in early preantral mouse follicle culture in vitro

In two consecutive controlled experiments 160 early preantral follicles were cultured in order to evaluate effects of recombinant follicle stimulating hormone (r-FSH) on survival, differentiation, oestradiol and inhibin secretion, cumulus mucification and cumulus-corona-oocyte detachment by human chorionic gonadotrophin (HCG) stimulation. Nuclear maturation in oocytes was also assessed following addition of HCG. A histological analysis of cultured follicles was carried out on semi- thin sections at various culture stages. Addition of r-FSH was essential for follicle survival for 16 days: without r-FSH only 11% of the follicles survived for 12 days (with r-FSH: 79%) and none of these mucified after the HCG stimulus. r-FSH promoted granulosa cell proliferation and antral-like cavity formation. Without r-FSH, histology of the cultures demonstrated degeneration and reduced granulosa cell proliferation; oestradiol and inhibin production were reduced. This study illustrates the essential role of FSH in promoting the in-vitro growth of early preantral mouse ovarian follicles and in maintaining the oocyte under meiotic arrest.      

Comparison of assisted reproductive technology performance after oocyte retrieval under general anaesthesia (propofol) versus paracervical local anaesthetic block: a case-controlled study

Propofol (Dipirivan) is an intravenous anaesthetic drug used for general anaesthesia. Although frequently used as a general anaesthetic for ultrasound procedures, its use during transvaginal oocyte retrieval is currently being debated. A total of 202 patients undergoing fertility treatment was included in a prospective, matched, controlled study, in which we compared fertilization rates and embryo development in terms of morphological quality and speed of development and the implications for reproductive outcome and pregnancy following general anaesthesia using either propofol or a paracervical local anaesthetic block during oocyte collection. There were no differences between the fertilization rates and the embryo cleavage characteristics for the two groups. The initial implantation rate per transferred embryo after general anaesthesia was similar to that after paracervical local anaesthetic block (13.4 versus 18.6%; P = 0.10). The ongoing clinical implantation rates per embryo transferred were also similar in the two groups.      

Timing of oocyte activation, pronucleus formation and cleavage in humans after intracytoplasmic sperm injection (ICSI) with testicular spermatozoa and after ICSI or in-vitro fertilization on sibling oocytes with ejaculated spermatozoa

In the first study, we evaluated 101 oocytes [2, 4, 6, 8, 16, 18 and 20 h after intracytoplasmic sperm injection (ICSI)] that had been microinjected with testicular spermatozoa. Of the 70 normally fertilized oocytes (69%) 30 (43%) had two pronuclei by 6 h after ICSI. Fifty-one (73%) by 8 h, 69 (99%) by 16 h and four of them by 20 h cleaved to the 2-cell stage. In the second study, 95 cumulus-corona- oocyte complexes (CCOC) were divided into two groups. Forty-seven CCOC were inseminated by conventional in-vitro fertilization (IVF) and 40 metaphase-II oocytes by ICSI. Oocytes were evaluated at 2, 4, 6 (only after ICSI), 8, 10, 12, 18, 20, 22, 24, 26, 28 and 30 h after both ICSI and IVF. After IVF, 35 oocytes were fertilized normally (75%), four of which (11%) had two pronuclei by 8 h, 11 (31%) by 10 h, 27 (77%) by 12 h and 35 (100%) by 14 h. The first cleavages had occurred by 24 h after insemination (four oocytes, 11%). After ICSI, 34 oocytes were fertilized normally (79%), 13 of which (38%) had two pronuclei by 6 h, 27 (79%) by 8 h and 32 (94%) by 10 h. Three oocytes cleaved by 20 h after microinjection (9%) and 19 by 24 h (56%). Pronuclei developed asynchronously in six oocytes after ICSI (18%) as opposed to 16 oocytes after IVF (46%). The results of this study suggest that the timing of pronuclear formation is no different when a testicular spermatozoon is microinjected into the oocytes from when an ejaculated spermatozoon is injected. Secondly, pronuclear development and first cleavage generally take place 4 h sooner after ICSI than after IVF. On the other hand, a higher proportion of oocytes develop two pronuclei asynchronously after IVF than after ICSI.      

The effect of female age and ovarian reserve on pregnancy rate in male infertility: treatment of azoospermia with sperm retrieval and intracytoplasmic sperm injection

Factors other than spermatozoa could be the major determinant of the success of assisted reproduction treatment in cases of male infertility. Our aim was to evaluate the effect of the wife's age and ovarian reserve on assisted reproduction success rates in the most severe type of male infertility, i.e. azoospermia. A total of 249 consecutive couples suffering from male infertility caused by azoospermia underwent microsurgical epididymal sperm aspiration (MESA) or testicular sperm extraction (TESE) with intracytoplasmic sperm injection (ICSI). Of these men, 186 had irreparable obstructive azoospermia, and 63 had non-obstructive azoospermia due to testicular failure. Neither the pathology, the source, the quantity, nor the quality of spermatozoa had any effect on fertilization or pregnancy rates. Maternal age and ovarian reserve (number of eggs) had no effect on fertilization or embryo cleavage, but did dramatically affect the embryo implantation, pregnancy and delivery rates. Wives of azoospermic men who were in their 20s had a 46% live delivery rate per cycle, wives aged 30-36 years had a 34% live delivery rate per cycle, wives aged 37- 39 years had a 13% live delivery rate per cycle, and wives > or = 40 years had only a 4% live delivery rate per cycle. The number of eggs retrieved also affected pregnancy and delivery rate, but to a lesser extent than age. In virtually all cases of obstructive azoospermia, and in 62% of cases with non-obstructive azoospermia caused by germinal failure, sufficient spermatozoa could be retrieved to perform ICSI, with normal fertilization and embryo cleavage. However, the pregnancy rate and the live delivery rate were dependent strictly on the age of the wife, and on her ovarian reserve. Unfortunately, exaggerated claims of high pregnancy rates can thus easily be made by manipulating, in a very simple way, selection for female factors.      

Molecular genetic and phenotypic analysis reveals differences between TSC1 and TSC2 associated familial and sporadic tuberous sclerosis

Tuberous sclerosis (TSC) is an autosomal dominant disorder characterised by the development of hamartomatous growths in many organs. Sixty to seventy percent of cases are sporadic and appear to represent new mutations. TSC exhibits locus heterogeneity: the TSC2 gene is located at 16p13.3 whilst the TSC1 gene, predicted to encode a novel protein termed hamartin, has recently been cloned from 9q34. With the exception of a contiguous gene deletion syndrome involving TSC2 and PKD1 , TSC1 and TSC2 phenotypes have been considered identical. We have now comprehensively defined the TSC1 mutational spectrum in 171 sequentially ascertained, unrelated TSC patients by single strand conformation polymorphism and heteroduplex analysis of all 21 coding exons, and by assaying a restriction fragment spanning the whole locus. Mutations were identified in 9/24 familial cases, but in only 13/147 sporadic cases. In contrast, a limited screen revealed TSC2 mutations in two of the familial cases and in 45 of the sporadic cases. Thus TSC1 mutations were significantly under-represented among sporadic cases (Fisher's exact p -value = 3.12 x 10(-4)). Both large deletions and missense mutations were common at the TSC2 locus whereas most TSC1 mutations were small truncating lesions. Mental retardation was significantly less frequent among carriers of TSC1 than TSC2 mutations (odds ratio 5.54 for sporadic cases only, 6.78 +/- 1.54 when a single randomly selected patient per multigeneration family was also included). No correlation between mental retardation and the type of mutation was found. We conclude that there is a reduced risk of mental retardation in TSC1 as opposed to TSC2 disease and that consequent ascertainment bias, at least in part, explains the relative paucity of TSC1 mutations in sporadic TSC.      

A stable, nonsense mutation associated with a case of infantile onset polycystic kidney disease 1 (PKD1)

Autosomal dominant polycystic kidney disease (ADPKD) is the most common single gene disorder resulting in renal failure. It is generally an adult onset disease, but rarely, cases of severe childhood polycystic disease arise in ADPKD families. The clear clinical anticipation in these pedigrees has led to the suggestion that the mutation may be an unstable trinucleotide repeat. We have now identified a nonsense mutation, Tyr3818Stop, in one such family (P117) within the major ADPKD gene, polycystic kidney disease 1 (PKD1). The mutation is shown to be a de novo change in the father, and of grandpaternal origin. PKD1 manifests as typical adult onset disease in the father, but is seen as severe disease, detected as enlarged polycystic kidneys in utero, in one of a pair of dizygotic twins; the other twin has the mutation but no evidence of cysts, consistent with an adult onset disease course. The finding of the same stable mutation associated with very different disease severity in this family indicates that phenotypic variation in PKD1 is not due to a dynamic mutation. It seems most likely that a small number of modifying factors may radically affect the course of disease in PKD1; identification of such factors will have important prognostic implications in this disorder.      

THERAPEUTIC BILIARY ENDOSCOPY: EXPERIENCE AT A SERVICE HOSPITAL

Therapeutic Biliary Endoscopy (TBE) is becoming a popular mode of treatment for patients with obstructive jaundice. This paper highlights our early experience of TBE at Armed Forces Medical College and Command Hospital (SC), Pune with this mode of treatment. TBE was used as a primary therapeutic option in 46 patients with obstructive jaundice. The age of the patients ranged from 11 to 80 (mean and SD:45.5 ± 16) years and majority 29 (63%) were males. The cause of obstructive jaundice in these patients was choledocholithiasis (n=31), benign biliary stricture (n=8), post cholecystectomy recurrent stones (n=3), carcinoma of pancreas (n=3) and papillary stenosis (n-1). Endoscopic Sphincterotomy (ES) was technically successful in all the 46 patients and brought prompt symptomatic relief in 43 patients. Sixteen patients (34.8%) required additional drainage such as stenting or nasobiliary drain. In patients with choledocholithiasis, bile duct could be cleared of stones in 29 (93.5%) patients and in two surgical removal was required. Of the remaining patients, surgery was required in 4 (50%) patients with benign biliary structure, in 1 (33.3%) of those with malignant stricture and none of the patients presenting with papillary stenosis or recurrent bile duct stones after cholecystectomy. Complications were seen in only two patients (4.4%): one had mild acute pancreatitis and another had GI bleed, which did not require blood transfusion. Both the complications were self-limiting. No procedure related deaths were noted. Endoscopic therapy, thus, a simple, effective and safe method of treatment in patients with choledocholithiasis and selected patients with malignant biliary obstruction.KEY WORDS: Biliary obstruction, Endoscopic sphincterotomy, Endotherapy, Gall stones, Obstructive jaundice, Therapeutic endoscopy     

SOLITARY RECTAL ULCER: CLINICO PATHOLOGICAL PROFILE OF A STUDY

Eighteen rectal biopsies of eleven patients were histologically diagnosed as solitary rectal ulcer (SRU) during a period of three years. Their clinical and sigmoidoscopic features were analysed. Correct clinical diagnosis of SRU was made in only two out of eleven cases, the rest were diagnosed after sigmoidoscopy and biopsy. Presenting features were protean predominated by an altered bowel habit or bleeding per rectum. Sigmoidoscopically the location of the lesion varied from 6 to 12 cm from the anal verge. Anterior rectal wall was more commonly affected (10 out of 11 cases). Single rectal ulcer was found in 7 patients, other 4 showing multiple ulcers or polypoidal non-ulcerative lesions. Three patients required multiple biopsies. Prominent histological findings were obliteration of lamina propria by fibromuscular strands, splaying and hypertrophy of muscularis musosae as well as infiltrate in both these layers. The necessity of biopsy to diagnose SRU is stressed.KEY WORDS: Rectal diseases, Sigmoidoscopy