Jehoshua Dor

Searching for evidence of disease and malignant cell contamination in ovarian tissue stored from hematologic cancer patients

BACKGROUND Storing ovarian tissue for fertility preservation in cancer patients carries the risk of the presence of malignant cells that could lead to recurrence of cancer after reimplantation. Methods to exclude presence of cancer cells were used to improve the safety of cryopreservation-reimplantation procedures. METHODS Fifty-eight patients with hematological malignancies were referred for the storage of ovarian tissue for fertility preservation. Investigation included preoperative imaging and histological evaluation of fresh ovarian tissue. After thawing markers to detect minimal residual disease (MRD) were used and compared with patients disease used as positive control (five patients). RESULTS Preoperative imaging detected disease in the ovaries (two patients). Conventional histology post-tissue harvesting did not disclose malignant cells (56 patients). MRD results post-thawing were negative in Hodgkins disease (CD30 immunohistochemical staining), in T- and B-cell lymphoma (PCR for T-cell receptor and Ig clones, respectively) and in two chronic myelogenous leukemia patients (RT-PCR for BCR-ABL gene expression). However, highly sensitive real-time RT-PCR was positive in one CML patient and, this alarming result avoided tissue transplantation. CONCLUSIONS Preoperative imaging prevented operations and storage of tissue with cancer. Evaluation of stored ovarian tissue for MRD using sensitive markers is essential to increase safety and to prevent reimplantation of tissue with malignant cells.

Cancer incidence in a cohort of infertile women who underwent in vitro fertilization

OBJECTIVE To assess whether ovarian hyperstimulation and IVF increase the risk for cancer. DESIGN Historical cohort analysis. SETTING; IVF units of two medical centers in Israel. PATIENT(S) Five thousand twenty-six women who underwent IVF between 1981 and 1992. INTERVENTION(S); Cancer incidence rates were determined through linkage to the National Cancer Registry and were compared with expected rates with respect to age, sex, and place of birth. MAIN OUTCOME MEASURE(S) Development of cancer. RESULT(S) Twenty-seven cases of cancer were observed, and 35.6 were expected (standardized incidence ratio, 0.76 [95% CI, 0.50-1.10]). Eleven cases of breast cancer were observed, whereas 15.86 were expected (standardized incidence ratio, 0.69 [95% CI, 0.46-1.66]). One case of ovarian cancer and 1 case of cervical cancer were observed, compared with 1.74 and 1.73 cases expected, respectively. The type of infertility, number of IVF cycles, and treatment outcome did not significantly affect risk for cancer. CONCLUSION(S) In a cohort of women treated with IVF, no excess risk for cancer was noted.

Congenital bilateral absence of vas deferens in the absence of cystic fibrosis

The high frequency of mutations in the cystic fibrosis gene in patients with congenital bilateral absence of vas deferens (CBAVD) has raised the question whether all of them have a genital form of cystic fibrosis. We investigated 47 CBAVD patients by ultrasonography, 10 (21%) had renal malformations and 37 (79%) did not. In the former group, no cystic fibrosis mutations were found and sweat chloride concentrations were normal. In the latter group, 18 patients (49%) carried at least one cystic fibrosis mutation and sweat chloride was high in 17 of 26 tested (65%). Our findings suggest that CBAVD patients with renal malformations do not necessarily have cystic fibrosis.

Sperm chromosome analysis and outcome of IVF in patients with non-mosaic Klinefelter’s syndrome

OBJECTIVE The aim of the study was to determine the potential risk for fetal chromosomal anomalies in non-mosaic Klinefelters syndrome patients undergoing IVF and intracytoplasmic sperm injection. DESIGN Individually collected spermatozoa were isolated from wet testicular tissue preparations and fixed on glass slides using micromanipulation. Their nuclei were analyzed for chromosomes X, Y, and 18 by fluorescent in situ hybridization. SETTING Assisted reproductive technology program. PATIENT(S) Consenting patients with non-mosaic Klinefelters syndrome undergoing testicular biopsy and IVF (fresh specimens) or following such treatment (cryopreserved specimens). INTERVENTION(S) None. MAIN OUTCOME MEASURE(S) The rates of numerical chromosome abnormalities for chromosomes X, Y, and 18 among spare testicular sperm and the pregnancy outcome following treatment. RESULT(S) Testicular sperm were found in 8 of 20 patients. Four couples became pregnant following embryo replacement. Sperm chromosomes were analyzed in five patients. One hundred and five sperm of 112 analyzed (93.7%) were normal with X to Y ratio of 50:55 (NS) respectively. Among the 112 sperm tested, seven (6.3%) demonstrated chromosomal abnormalities, of which five were related to the sex chromosomes and two to chromosome 18. One set of triplets, one set of twins, and two singletons (four males and three females) with normal karyotypes were born. CONCLUSION(S) Most of the testicular sperm retrieved from Klinefelters syndrome patients demonstrates a normal pattern of sex chromosome segregation. Therefore, the risk of transmitting numerical sex chromosome abnormalities is relatively low and probably comparable with the rates found in other severe male factor infertility patient groups.

Sperm chromosome abnormalities in men with severe male factor infertility who are undergoing in vitro fertilization with intracytoplasmic sperm injection

OBJECTIVE To investigate the potential paternal contribution to the risk of fetal chromosomal anomalies after intracytoplasmic sperm injection (ICSI). DESIGN Spermatozoa isolated from testicular tissue and ejaculated specimens of consenting patients undergoing testicular biopsy and ICSI were analyzed for chromosomes X, Y, and 18 by FISH. SETTING Assisted reproductive technology program. PATIENT(S) Consenting patients undergoing testicular biopsy and ICSI, severe oligozoospermic patients, and normal fertile donors. INTERVENTION(S) None. MAIN OUTCOME MEASURE(S) The rate of chromosome abnormalities in testicular sperm with regard to the type of azoospermia and ejaculated sperm compared to healthy men. RESULT(S) The mean serum levels of FSH in the groups with nonobstructive azoospermia (n = 9), obstructive azoospermia (n = 10), severe oligozoospermia (n = 9), and the normal donors (n = 6) were 17.5 +/- 8.2 (P<.05), 3.5 +/- 2.6, 14.6 +/- 3.5 (P<.05), and 3.1 +/- 0.4 IU/mL, respectively. The corresponding rates of sperm chromosome abnormalities among these groups were 19.6% (P<.001), 8.2% (P<.001), 13.0% (P<.001), and 1.6%, respectively. The corresponding rates of disomy among these groups were 7.8% (12 of 153 spermatozoa), 4.9% (18 of 367), 6.2% (109 of 1,751), and 1% (5 of 500 spermatozoa), respectively. Errors in chromosomes X and Y were significantly more common than in chromosome 18. CONCLUSION(S) The present findings demonstrate a linkage between gonadal failure (high serum FSH levels) and the occurrence of sperm chromosome aneuploidies. Our findings may explain the increased incidence of sex chromosome abnormalities found after IVF in the severe male factor patient population. Genetic screening during pregnancy or before embryo replacement should be considered carefully.

Aging of endometrium and oocytes : observations on conception and abortion rates in an egg donation model

OBJECTIVE To assess the comparative contribution of endometrial and oocytic aging to the decline in fertility with age. DESIGN Retrospective analysis of conception and abortion rates in an egg donation program, with respect to donor and recipient ages. PATIENTS All oocyte recipients had ovarian failure. Donors were women undergoing in vitro fertilization who contributed up to a third of retrieved oocytes. INTERVENTIONS None. RESULTS Thirty pregnancies (28 intrauterine) were recorded in 169 reception cycles originating from 91 donation cycles. Women who conceived were younger than those who did not (median age 31 versus 37; P less than 0.046), with no difference in age of donors. There was a significant difference in spontaneous abortion rate by age of donor rather than by age of recipient. Donors to successful pregnancies were younger than donors to aborted pregnancies (median age 27.5 versus 33; P less than 0.0211), but the 11 women with aborted pregnancies did not differ in age from the 17 women with successful pregnancies. CONCLUSIONS In women, endometrial function as expressed by conception rate in the recipients declines with age, whereas it is oocyte age that primarily influences risk of abortion.

Cumulative conception rates following gonadotropin therapy

During the years 1963 to 1978, 515 patients were treated with HMG. They were divided into two groups depending upon the absence (Group I) or presence (Group II) of distinct endogenous estrogen activity. Group II patients were referred after failure of clomiphene therapy. None of the patients had mechanical or male infertility factors. The cumulative pregnancy rate (life table method) in Group I patients after six cycles of treatment was 91.2%. In 77 patients from Group I, further treatment was given for a second pregnancy after the first gonadotropin conception. In this group, the cumulative conception rate was 93.6% after eight cycles of treatment. In Group II the cumulative conception rate was 50% after 12 cycles of treatment. In both groups, the results were better in patients who were less than 35 years of age than those who were 35 or more when treated.

The incidence of combined intrauterine and extrauterine pregnancy after in vitro fertilization and embryo transfer

Our data demonstrate the high incidence of combined pregnancy among patients who have undergone IVF. Close monitoring of pregnancies in IVF programs may prevent a possibly deleterious delay in the treatment of simultaneous ectopic and intrauterine gestation.

Chromosome analysis of multipronuclear human oocytes fertilized in vitro

In an in vitro fertilization and embryo transfer program composed mainly of patients with tubal infertility, the incidence of fertilized oocytes with multiple pronuclei was 4.3%. An attempt was made to fix such oocytes with supernumerary pronuclei in order to assess the chromosome constitution of the gametes. Nine multipronuclear oocytes containing a total of 29 pronuclei were successfully fixed before the first cleavage division, and another oocyte containing 3 pronuclei was fixed before the second cleavage division. On analysis, chromosome counts could be obtained for 28 of the 29 total pronuclei, and 24 gave informative results. Nineteen pronuclei had a normal haploid chromosome count, 2 pronuclei contained one extra chromosome, and 3 pronuclei had one chromosome missing. The presence of a Y chromosome in six pronuclei identified their paternal origin; two of the six sperm pronuclei had a 22,Y,-E chromosome constitution. Only three pronuclei could be conclusively ascertained to be maternal in origin, yet two of these were aneuploid; one pronucleus had a 24,X,+D karyotype and the second had only 22 chromosomes. Multipronuclear oocytes present ideal material for analyzing the chromosome constitution of those human gametes which can undergo fertilization in vitro.

Prognostic value of the clinical and laboratory evaluation in patients with nonmosaic Klinefelter syndrome who are receiving assisted reproductive therapy

OBJECTIVE To characterize clinical and laboratory findings in nonmosaic 47,XXY patients that may help to predict spermatogenetic activity in their testicles. DESIGN Prospective study. SETTING Assisted reproductive technology program. PATIENT(S) Twenty patients with nonmosaic Klinefelter syndrome who underwent testicular sperm retrieval for IVF. MAIN OUTCOME MEASURE(S) The correlation between basal FSH, LH and testosterone levels, mean testicular volume, and results of the hCG test and presence or absence of sperm after testicular sperm extraction (TESE). RESULT(S) Sperm was found in nine patients (45%). The mean testicular volume was 7.8 +/- 2.5 mL in men with sperm after TESE and 5.6 +/- 1.2 mL in those without sperm after TESE; corresponding testosterone levels were 3.5 +/- 1.2 ng/mL and 1.7 +/- 0.8 ng/mL. Serum levels of FSH and LH did not significantly differ between groups. After the hCG test, the mean serum testosterone level was 16.0 +/- 6.3 ng/mL in men with sperm after TESE and 6.7 +/- 5.6 ng/mL in those without sperm. CONCLUSION(S) Testicular volume, testosterone levels, and results of the hCG test are important predictive factors of spermatogenesis in patients with nonmosaic Klinefelter syndrome.