Preimplantation genetic diagnosis of chromosomal abnormalities by multicolour fluorescence in situ hybridisation.

Preimplantation genetic diagnosis (PGD) is an early diagnosis of genetic disorders, prior to the onset of pregnancy. PGD incorporates the latest techniques in assisted reproduction and molecular genetics. Embryos or oocytes are biopsied during culture in vitro and genetic analysis is carried out on the blastomeres or polar bodies. Embryos shown to be free of the genetic disease under investigation are transferred to the uterus. Multicolour fluorescence in situ hybridisation (FISH) is used to diagnose numerical and certain structural abnormalities of chromosomes in the embryo. The common probes used are for chromosomes 13, 18, 21, X and Y. FISH can also be used for PGD of translocations, when one of the parents is a carrier. PGD was carried out recently in 4 cases using multicolour FISH. In one of the embryos, trisomy 18 was detected. Tetraploidy was seen in another embryo. Only chromosomally normal embryos were transferred back to the uterus. Care has to be taken while interpreting FISH signals as the signal may be split, diffused, superimposed or in a different focus.

Coping mechanisms in patients presenting for in-vitro-fertilization.

OBJECTIVES: The purpose of this study was to assess the coping mechanisms in patients presenting for in-vitro fertilization (IVF). METHODS: We evaluated thirty consecutive couples presenting for in-vitro-fertilization. All couples were interviewed individually at first, and then together, using a semi-structured interview technique. Psychiatric diagnoses were made using the Diagnostic and Statistical Manual-IV (DSM-IV) criteria. Coping mechanisms used by the individuals were assessed using the Mechanisms of Coping Scale (MOCS). Other instruments used were Hamilton Depression Rating Scale (HAM-D-17), Hamilton Anxiety Rating Scale (HAM-A), Brief Psychiatric Rating Scale (BPRS), Self-Rating Symptom Scale (SRSS), and Eysenck Personality Inventory (EPI). RESULTS: The mean age of the sixty patients was 32.3 +/- 5.2 years. Fatalism was the commonest factor on the mechanisms of coping scale. Analysis of variance (ANOVA) across all factors of the MOCS for demographic factors showed that men used problem-solving mechanisms significantly more often than women (F = 3.0, df = 1, 58, p < 0.05). ANOVA across coping factors on stressors with post-hoc tests of significance revealed that individuals facing social stress used fatalism significantly more often than other coping mechanisms, while those facing career stress used problem-solving significantly more often than other coping mechanisms (F = 5.6, df = 1, 58, p < 0.05 and F = 3.04, df = 1, 58, p < 0.01 respectively). ANOVA across coping factors on HAM-D-17 scores revealed that individuals who used fatalism had significantly higher HAM-D-17 scores compared to those who did not (F = 4.4, df = 1, 58, p < 0.05). ANOVA across coping factors on HAM-A scores revealed that individuals who used escape-avoidance had significantly lower HAM-A scores than those who did not (F = 4.3, df = 1, 58, p < 0.05). ANOVA across coping factors on SRSS scores revealed that individuals who used passivity or fatalistic coping mechanisms had significantly higher scores on SRSS than who did not (F = 4.6, df = 1, 58, p < 0.05 and F = 3.5, df = 1, 58, p < 0.05). CONCLUSIONS: Differential patterns of coping were found among the sixty individuals presenting for IVF and were associated with a variety of factors including gender, education, stressors, and levels of depression, anxiety, and overall psychopathology. Efforts to recognize and recruit the coping mechanisms of infertile individuals are likely to enhance their ability to participate effectively in treatment.

Diagnostic significance of immunoglobulins and adenosine deaminase in pleural effusion.

OBJECTIVES: 1) To evaluate the diagnostic significance of ELISA for detection of antibodies to antigen A60 of Mycobacterium tuberculosis and adenosine deaminase (ADA) in patients with pleural effusion without prior history of tuberculosis, 2) To study the importance of the above tests in patients with HIV infection and pleural effusion. METHOD: Eighty one patients with pleural effusion were studied. In addition to diagnostic paracentesis and pleural biopsy, pleural fluid specific IgG, IgM, IgA against antigen A60 and fluid ADA were estimated. HIV testing (ELISA) was done in all patients. RESULTS: Out of 81 patients, 13 were HIV positive. IgG anti A60 had a sensitivity and specificity of 90.71% and 33.33% respectively. The combination of IgG and IgM had a sensitivity and specificity of 96.3% and 55.55% respectively. IgA alone or in combination did not offer any diagnostic advantage. ADA had a sensitivity and specificity of 76% and 60% respectively. In presence of HIV infection, antibodies against A60 had a sensitivity of 76.92% and ADA had a sensitivity of 61.54%. CONCLUSIONS: In patients with pleural effusion, immunoglobulins against antigen A60 have a limited diagnostic role with high false positive rates. Co infection with HIV further reduces the value of above diagnostic tests.

Genital tuberculosis--a major pelvic factor causing infertility in Indian women.

OBJECTIVE: To study the effect of tuberculosis, a common infectious disease in the Indian subcontinent, on the female pelvic factor and its subsequent effect on female fertility. DESIGN: Retrospective case studies. SETTING: Department of Infertility Management and Assisted Reproduction, Jaslok Hospital and Research Centre, Bombay, India. PATIENT(S): Three hundred women, between the ages of 25 and 35 years, with tubal factor as a cause of their infertile state. RESULT(S): One hundred seventeen women with a tubal factor were found to have tuberculosis as the cause of tubal blockage. On laparoscopy, 49.5% were found to have simple tubal blockage, 15.3% showed tubo-ovarian masses, and 23.9% had a frozen pelvis. Seventy-five percent complained of menstrual irregularities, thus indicating endometrial involvement; 25.6% of these women underwent an IVF procedure. The pregnancy rate after IVF-ET was 16.6% per transfer. CONCLUSION(S): This study highlights the fact that tuberculosis, a chronic infectious disease, is one of the major etiologic factors of female tubal infertility, especially on the Indian subcontinent.

Computer-assisted semen analysis parameters in men with varicocele: is surgery helpful?

OBJECTIVE: To assess sperm characteristics and fertility before and after varicocelectomy using computer-assisted semen analysis (CASA). DESIGN: Preoperative and postoperative sperm parameters of infertile men with varicocele were analyzed statistically and the outcome of pregnancy was determined. SETTING: Department of Infertility Management and Assisted Reproduction, Jaslok Hospital and Research Centre, Bombay. PATIENTS: Semen samples of 49 men with varicocele-related infertility were analyzed using CASA. INTERVENTIONS: Sperm parameters of 26 men who underwent varicocelectomy were evaluated 3, 6, and 9 months postoperatively. Conception, occurring either naturally or therapeutically, was recorded. MAIN OUTCOME MEASURES: Evaluation of improvement of sperm parameters after surgery. RESULTS: Sperm count, motility parameters, curvilinear velocity, straightline velocity, lateral head displacement, and normal morphology were significantly lower in men with varicocele. Postoperatively, there was significant improvement in count, motility, and normal morphology, with a decrease in proportion of acrosome-deficient heads and tapering forms. After varicocelectomy, 46.2% of the men had normal semen parameters, with the overall pregnancy rate being 50%. CONCLUSIONS: Computer-assisted semen analysis provides the potential for accurate quantitative evaluation of semen in men with varicocele. Varicocelectomy results in improvement in semen quality with pregnancy rates of 50%.

The importance of computer-assisted semen analysis and sperm function testing in an IVF program.

OBJECTIVE: To study the relationship of sperm motion characteristics, sperm morphology, and hypo-osmotic swelling test with fertilization rates in vitro. DESIGN: Computer-assisted measures of fresh seminal and processed sperm preparations, sperm morphology, and hypo-osmotic swelling test results were assessed for predicting fertilization by step-wise regression analysis. SETTINGS: In vitro fertilization laboratory, department of infertility management, university affiliated-hospital. INTERVENTIONS: None. PATIENTS: One hundred and two couples who underwent IVF were studied. MAIN OUTCOME MEASURES: Computer-assisted sperm motion variables in semen and following semen processing in capacitating media; hypo-osmotic swelling of sperm tails before and after semen processing; morphology of sperm before and after processing; fertilization of oocytes as evidenced by presence of two pronuclei as the end point. RESULTS: Various sperm motion parameters, hypo-osmotic swelling test results, and normal morphology of sperm were directly correlated to fertilization as judged by the Karl-Pearsons test. However, in step-wise regression analysis, normal morphology of sperm from seminal fraction exhibited 61% correlation with fertilization rates (P < .001). In step 2, normal morphology along with percent motility exhibited a 64% correlation with fertilization rates (P < .001). In step 3, normal morphology, percent motility, and linearity showed a 67% correlation with fertilization rates (P < .001). The hypo-osmotic swelling test did not predict fertilization rates (regression coefficient = 0.066, P = 0.474). In the processed sample preparations, normal sperm morphology showed a 72% correlation with fertilization rate (P < .001). In step 2, normal morphology along with curvilinear velocity exhibited a 77% correlation with fertilization rates (P < .001). In step 3, normal morphology, curvilinear velocity, and average path velocity showed a 79% correlation with fertilization rates (P < .001). The hypo-osmotic swelling test did not predict fertilization rates (regression coefficient = 0.076, P = 0.512). CONCLUSIONS: Morphology of sperm and computer-assisted sperm motion variables, such as motility, linearity, curvilinear velocity, and average path velocity, may serve as prognostic indicators for fertilization potential of sperm. The hypo-osmotic swelling test may describe only physiological intactness, rather than the fertilization potential of sperm. The results suggest that objective analysis of sperm motion characteristics and precise sperm morphology may form a first, and obligatory, step for critical evaluation of patients before they start IVF treatment.

Assisted hatching in an in vitro fertilization programme.

Assisted hatching was performed on 37 women (IVF, n = 12; micromanipulation, n = 25), who were at risk of poor implantation after in vitro fertilization and micromanipulation, and who had previous cycles of failed implantation. A total of 292 oocytes was retrieved (7.9 per cycle) of which 90 were subjected to IVF and 202 were micromanipulated. Assisted hatching was performed on day 3 of culture, on six- to eight-cell embryos, by the technique of mechanical partial zona dissection. All the patients undergoing the procedure of assisted hatching had embryos with thick zonae, greater than 15 microns. Assisted hatching was performed on 39 embryos (55.7%) in the IVF group and 104 embryos (77.6%) in the micromanipulation group. The damage rate after hatching was 1.4%. The pregnancy rate per cycle was 50% in the IVF group and 44% in the micromanipulation group, compared with 10% (IVF) and 19.2% (micromanipulation) in the control groups. Assisted hatching, by partial zona dissection, results in significant improvement in pregnancy rates in women more than 38 years of age, who have a thick zona and with previous poor implantation results. This paper reports the first pregnancy in India by the technique of assisted hatching (partial zona dissection).

Multinuclear spermatozoa associated with polyploidy.

We are reporting on a possible association between multinucleated spermatozoa and polyploidy after in vitro fertilization. The semen from the male partner of a couple with 40% polyploidy on three in vitro fertilization attempts was examined by light and transmission electron microscopy. In that sample 10% to 12% of spermatozoa demonstrated conjoined or double heads. The significance of these findings is discussed.

Measurement of CA125, carcinoembryonic antigen, and alpha-fetoprotein in ovarian cyst fluid: diagnostic adjunct to cytology.

This study used CA125, carcinoembryonic antigen (CEA), and alpha-fetoprotein (AFP) to classify ovarian cysts by measuring the levels of the three antigens; this information was useful when fluid obtained through laparoscopic puncture of ovarian cysts was submitted for cytologic examination from patients for whom tissue was unavailable for classification. We studied 136 consecutive cyst fluids (108 benign, 28 malignant) and correlated the findings with the tissue diagnosis. All three antigens were very low (CEA, less than 0.5 ng/ml; CA125, 55-2,143 mu/ml; and AFP, less than 4.8 ng/ml) in follicular and lutein cysts. Markedly elevated CA125 (296-1,950,000 mu/ml) and low CEA (0.5-220 ng/ml) and AFP (less than 4.8 ng/ml) levels were seen in patients with serous neoplasms, both benign and malignant. Elevated CEA (greater than 600 ng/ml) and CA125 (56-65,330 mu/ml) levels were seen in primary mucinous cystadenoma and cystadenocarcinoma. Two patients with colonic carcinoma metastatic to the ovary had an elevated CEA (greater than 600 ng/ml) and a normal CA125. Only one patient, with a malignant teratoma, had an elevated AFP. The adjunctive use of CEA and CA125 is recommended for the classification of ovarian cysts.