Preimplantation diagnosis by fluorescence in situ hybridization using 13-, 16-, 18-, 21-, 22-, X-, and Y-chromosome probes.

PURPOSE: Our purpose was to select the proper chromosomes for preimplantation diagnosis based on aneuploidy distribution in abortuses and to carry out a feasibility study of preimplantation diagnosis for embryos using multiple-probe fluorescence in situ hybridization (FISH) on the selected chromosomes of biopsied blastomeres. METHODS: After determining the frequency distribution of aneuploidy found in abortuses, seven chromosomes were selected for FISH probes. Blastomeres were obtained from 33 abnormal or excess embryos. The chromosome complements of both the biopsied blastomeres and the remaining sibling blastomeres in each embryo were determined by FISH and compared to evaluate their preimplantation diagnostic potential. RESULTS: Chromosomes (16, 22, X, Y) and (13, 18, 21) were selected on the basis of the high aneuploid prevalence in abortuses for the former group and the presence of trisomy in the newborn for the latter. Thirty-six (72%) of 50 blastomeres gave signals to permit a diagnosis. Diagnoses made from biopsied blastomeres were consistent with the diagnoses made from the remaining sibling blastomeres in 18 embryos. In only 2 of 20 cases did the biopsied blastomere diagnosis and the embryo diagnosis not match. CONCLUSIONS: If FISH of biopsied blastomere was successful, a preimplantation diagnosis could be made with 10% error. When a combination of chromosome-13, -16, -18, -21, -22, -X, and -Y probes was used, up to 65% of the embryos destined to be aborted could be detected.

Sex determination by simultaneous application of polymerase chain reaction and fluorescent in situ hybridization on the same blastomere of a pre-embryo.

OBJECTIVE: To describe an investigation of the feasibility of carrying out polymerase chain reaction (PCR) and fluorescent in situ hybridization on the same single human cell for sex determination. DESIGN: To develop protocol for a clinical diagnostic test in preclinical trials. SETTING: Infertile human volunteers in a clinical environment. PATIENTS: Polyploid embryos were obtained from patients treated by IVF at the Advanced Institute of Fertility. INTERVENTIONS: Seventeen blastomeres biopsied from human polyploid embryos were analyzed for sex determination by simultaneous application of PCR and fluorescent in situ hybridization. MAIN OUTCOME MEASURE: Presence of an X- or X and Y-chromosome band after agarose gel electrophoresis of PCR products and presence of an X- or X and Y-chromosome fluorescent probe signal after fluorescent in situ hybridization following PCR analysis. RESULTS: By PCR, all 17 blastomeres were amplified and, by fluorescent in situ hybridization, 12 (70.6%) blastomeres produced signals that were consistent with PCR results. Two blastomeres showed only X signals, although by PCR they had both X and Y-chromosome bands. CONCLUSIONS: The sequential use of PCR and fluorescent in situ hybridization on the same blastomere can be applied to improve the accuracy of sex determination before fresh ET.

Failed oocyte retrieval after lack of human chorionic gonadotropin administration in assisted reproductive technology.

OBJECTIVE: To document the absence of oocytes in follicular aspirates in women who, during controlled ovarian stimulation with gonadotropin-releasing hormone agonist (GnRH-a) and menotropins, fail to receive human chorionic gonadotropin (hCG) administration. DESIGN: Retrospective analysis of clinical laboratory data. SETTING: Multicentric. PATIENTS: Five women undergoing controlled ovarian hyperstimulation with GnRH-a and menotropins for programs of assisted reproductive technologies. RESULTS: The documented absence of an hCG injection produced "empty follicles" at transvaginal guided aspiration, despite numerous follicular lavages and aspiration of peritoneal fluid. The lack of oocytes and granulosa-cumulus complex in the follicular fluid was reverted in other cycles in the same patients when hCG was properly administered. CONCLUSIONS: (1) This study emphasizes the importance of proper patients' and nurses' instructions for preparation of hCG injections and proper mixture of vehicle and powder before follicular aspiration. (2) In the absence of cumulus-corona-oocyte complex at aspiration, measure serum beta-hCG to ascertain whether hCG injection was administered or not. (3) Routine preoperative beta-hCG levels may be helpful to avoid unnecessary surgeries.

Cryopreservation of human embryos as a useful tool for infertile couples.

In order to improve the pregnancy rate per oocyte retrieval while reducing the chance of multiple-gestations, a technique of cryopreservation of human embryos was used for our initial study consisting of 100 infertile patients undergoing in vitro fertilization (IVF) procedure. These patients combined a "fresh" embryo transfer with cryopreservation. The embryos were cryopreserved with 1,2-propanediol. Twenty-nine (29%) patients achieved ongoing pregnancies from the fresh embryo transfer. Thirty patients received a transfer of thawed embryos in a natural cycle. Nine of these patients achieved ongoing pregnancies. Combining these 2 transfers results in a 38% ongoing pregnancy rate. Forty-one patients have yet to receive their frozen embryos. The current success rate in this study indicates that over 50% of the patients in this group can expect to become pregnant by the time all the cryopreserved embryos have been transferred.

Analysis of false negative cytology by a chromosome study.

Forty-two cases of pre-invasive and invasive cancer of the cervix diagnosed by histology were studied cytologically and cytogenetically. Preinvasive lesions showed a good correlation between cytologic impression and the histologic diagnosis. However, among 17 patients with invasive cancer, there were 2 completely negative cytologic impressions. These were in cases having modal chromosome counts of 47 and 48, respectively. This study demonstrates that some invasive cancers of the cervix are resistant to cytologic diagnosis because of their peridiploid chromosome counts and lack of anisokaryosis.

Treatment of human spermatozoa with an egg yolk medium can enhance the outcome of in vitro fertilization.

In at least 4 of 7 cases, fertilization of intact human oocytes was more successful when spermatozoa were pretreated with TEST yolk medium at 5 degrees C for 2 hours as compared with the standard treatment with Ham's F-10 only. Both pregnancies that were obtained after the transfer of the fertilized oocytes resulted from oocytes fertilized by TEST yolk-treated spermatozoa. No decrease in fertilization occurred in any of the cases after TEST yolk treatment. If these results hold true for a larger series of patients, it may be worthwhile for the standard IVF incubation system of spermatozoa to include TEST yolk.

Karyotype analysis of blighted ova in pregnancies achieved by in vitro fertilization.

Pregnancies achieved by IVF and resulting in blighted ovum were karyotyped. The rate of aneuploidy was found to be 38% (5/13). In vitro fertilization does not appear to be a risk factor for chromosome anomaly in live births or miscarriages, and the data reported so far may suggest that aneuploid rates are actually lower for pregnancies achieved by IVF.

Short-term use of gonadotropin-releasing hormone agonist (leuprolide) for in vitro fertilization.

A common problem encountered by in vitro fertilization (IVF) programs is the premature occurrence of the spontaneous luteinizing hormone (LH) surge during ovarian stimulation cycles. Administration of gonadotropin-releasing hormone agonists (GnRH-a) for 2 to 3 weeks produces a state of hypogonadotropic hypogonadism, thus allowing ovarian stimulation to proceed uncomplicated by a spontaneous LH surge. We have elected to treat seven patients with GnRH-a in a "short-term" protocol, with GnRH-a initiated on cycle day 3 along with exogenous gonadotropins. In this series, we found that the spontaneous LH surge was abolished, while ovarian responsiveness seemed to be improved. These results suggest that the initial surge of gonadotropins elicited by GnRH-a administration may enhance ovarian stimulation and that spontaneous LH surge is blocked when GnRH-a and exogenous gonadotropins are initiated concomitantly.

Ultrasound-guided transvaginal needle aspiration of follicles for in vitro fertilization.

In search of a simpler and less traumatic method of oocyte retrieval for in vitro fertilization (IVF), 37 infertile patients underwent ultrasound-guided transvaginal needle aspiration of the follicles instead of the conventional laparoscopic aspiration. Although only intravenous analgesics and sedatives were used, patients experienced little discomfort. There were no infections or bleeding complications after the procedure. Eleven patients (30%) achieved ongoing pregnancies; this rate of success is comparable to that with our laparoscopic method. Considering this reasonable pregnancy rate, lack of substantial morbidity, and good patient acceptance, laparoscopic oocyte retrieval for IVF can be largely replaced by the ultrasound-guided transvaginal needle aspiration technique.

Five hundred cases of amniocentesis without bloody tap.

Five hundred amniocenteses were carried out at midtrimester for prenatal diagnosis. A 22-gauge spinal needle with a stylet was used. The needle was guided by a real-time sector ultrasonograph. A transducer was placed adjacent to the puncture site and the needle tip was observed entering the amniotic cavity. By this technique, a bloody tap was avoided. There were only three (0.6%) spontaneous abortions in this group of 496 patients. Amniocentesis guided by ultrasound is a safe method for the mother and the fetus.

Gonadoblastoma in a patient with an isodicentric X chromosome.

Although gonadoblastoma is known to be associated with the presence of a Y chromosome, this case report of a 15-year-old patient with gonadal dysgenesis, gonadoblastoma, and an idic(Xq-) chromosome provides evidence for the occurrence of gonadoblastoma even in the absence of a Y chromosome. A review of previous cases reported to have gonadoblastoma in the absence of a Y chromosome revealed that the presence of some breast development was the common denominator among those patients. Therefore, a patient who presents with gonadal dysgenesis with some breast development, but lacks a Y chromosome may observe as close scrutiny as the patient with gonadal dysgenesis in the presence of a Y chromosome.

Estrogen therapy for gonadal dysgenesis: a rational approach.

The administration of estrogens for gonadal dysgenesis is sometimes associated with the development of endometrial neoplasms. An approach which minimizes the inadvertent side effects while still providing the desired therapeutic effects may be to imitate the hormonal milieu of the normal menstruating woman. Oral administration of estradiol will not necessarily accomplish this, because of the intestinal conversion of estradiol to estrone. That problem can be overcome by the vaginal administration of estrogens.

Clinical delineation of trisomy 9 syndrome.

A rare but typical case of trisomy 9 mosaicism is reported. The infant showed the characteristic phenotype of this syndrome: low-set malformed ears, micrognathia, broad nose with bulbous tip, abnormal brain, congenital heart disease, abnormal hands and feet, cryptorchidism, micropenis, and early death. This infant, however, did not have dislocation of joints or microcephaly; the head was larger than normal and had almost a cloverleaf shape.

Advances in infertility.

PIP: Recent advances in the diagnosis and treatment of infertility, including in vitro fertilization, the use of danazol for endometriosis, and the availability of bromocriptine for the galactorrhea-amenorrhea syndrome, are described. The most common sequence for infertility work-up is: 1) evaluation of basal body temperature, 2) postcoital testing, 3) semen analysis, 4) endometrial biopsy, 5) hystersalpingography and Rubin's test, and 6) endoscopy. When this type of organized investigation reveals the causal factors responsible for infertility, therapy can be instituted. Treatment can focus around ovulatory disorders, cervical problems, male factors, luteal phase defects, tubal factors, uterine factors, or endometriosis. Objective comparison of the various treatments through the establishment of standardized classifications and organized regimens is important to future fertility investigation. A problem in comparing the pregnancy rates of treatments has been the variability in length of follow-up. This difficulty has been largely overcome by the method for calculating the "expectancy" of pregnancy with an assumption of indefinite follow-up.^ieng