Uncomplicated pregnancy and normal singleton delivery after surgical excision of heterotopic (cornual) pregnancy following in vitro fertilization/embryo transfer.

A 39 year-old woman with previous salpingectomy developed a symptomatic heterotopic right cornual pregnancy identified by transvaginal ultrasonography at six weeks' gestation. The patient had previously undergone an ipsilateral partial salpingectomy, and the conception was established four months later after one cycle of controlled ovarian hyperstimulation, in vitro fertilization (IVF) and embryo transfer. We performed immediate surgical excision of the ectopic implantation with conservation of the intrauterine pregnancy. Progesterone was administered as 200 mg/d lozenge (troche) plus 200 mg/d rectal suppository, maintained from day of embryo transfer through the perioperative period and until 11th gestational week. Following an uneventful obstetrical course, a healthy male infant was delivered by cesarean at term. In this report, we review the incidence and significance of heterotopic gestation in the context of IVF/embryo transfer. Risk factors for complex intra- and extra-uterine pregnancies are also outlined. Additionally, the clinical management of heterotopic pregnancy, including a novel approach to progesterone supplementation, is discussed.

Clinical application of human egg cryopreservation.

Clinical egg cryopreservation has been applied during a 4-year period with some limited success. Mostly mature and a few immature eggs were frozen slowly and thawed rapidly in 1,2-propanediol and sucrose, and subsequently inseminated by intracytoplasmic sperm injection (ICSI). Three studies were performed in which: (i) it was established that 55% of aged unfertilized mature eggs survive freezing; (ii) in 22 cycles of thawed donated eggs cryosurvival was 24% with 15 cycles reaching transfer, and five pregnancies were initiated, one of which went to term at 39 weeks with fraternal twin boys, and one remains ongoing at 37 weeks; and (iii) in five cycles, where in-vitro fertilization patients had some of their own eggs frozen/ thawed, cryosurvival of mature eggs was poor at only 2.2%, although 44% sibling germinal vesicle (GV) stage eggs survived. A normal female infant delivered at 40 weeks arose from transfer of two embryos where GV eggs underwent in-vitro maturation post-thaw and were fertilized by ICSI. Pregnancies reported here and by others indicate a burgeoning awareness of the potential benefits of egg cryopreservation, prompting cautious optimism for the future of this technology.

Birth after cryopreservation of immature oocytes with subsequent in vitro maturation.

OBJECTIVE: To establish the clinical feasibility of using cryostored germinal vesicle oocytes for IVF and ET. DESIGN: Case report. SETTING: Private infertility clinic. PATIENT(S): A 28-year-old woman with tubal infertility undergoing IVF therapy. INTERVENTION(S): Oocytes collected after ovarian stimulation were frozen without insemination or were inseminated, fertilized, and frozen as cleavage stage embryos. No fresh oocyte or embryo transfer was undertaken. All oocytes were thawed, and those that survived were used for IVF-ET. MAIN OUTCOME MEASURE(S): Oocyte cryosurvival, in vitro maturation, fertilization, embryo development, and pregnancy outcome. RESULT(S): None of 16 mature oocytes survived thawing; however, three of 13 germinal vesicle oocytes survived. After 30 hours in vitro maturation two oocytes had matured and underwent intracytoplasmic sperm injection with the partner's sperm. Both fertilized normally and were transferred to the patient. The woman delivered an apparently healthy female infant at 40 weeks. CONCLUSION(S): This case report proves the feasibility if not the efficiency of using immature oocytes for cryostorage, coupling both cryopreservation and in vitro maturation.

Reinsemination by intracytoplasmic sperm injection of 1-day-old oocytes after complete conventional fertilization failure.

OBJECTIVE: To analyze the efficacy of intracytoplasmic sperm injection (ICSI) for "rescue" of failed conventional insemination cycles. DESIGN: Retrospective clinical study. SETTING: Private infertility clinic. PATIENT(S): Fifty-four couples with non-male factor infertility undergoing routine IVF within our clinic. INTERVENTION(S): Twenty- to 24-hour-old unfertilized mature oocytes from conventional IVF cycles in which fertilization failure was complete were reinseminated by ICSI to attempt late fertilization and rescue of the otherwise failed IVF cycle. MAIN OUTCOME MEASURE(S): Late fertilization, pregnancy, and embryonic implantation. RESULT(S): Intracytoplasmic sperm injection reinsemination of 489 unfertilized mature oocytes caused degeneration in 50 (10.2%) oocytes, gave rise to normal fertilization in 215 (44.0%) of the injected oocytes, and allowed ET in 48 of the 54 cycles in which initial complete fertilization failure had occurred. One hundred sixty-four (76.3%) of 215 late-fertilized oocytes either were used for fresh transfer or were frozen-stored. Eight viable pregnancies resulted, yielding a 14.8% pregnancy rate per initiated cycle. CONCLUSION(S): Barring potential concerns regarding the chromosomal normality of embryos arising from reinsemination, our results suggest that ICSI is a relatively successful means of rescuing conventional IVF cycles in which fertilization fails completely.

Multiple pregnancy rate and embryo number transferred during in vitro fertilization.

OBJECTIVE: Our goal was to achieve a good pregnancy rate after in vitro fertilization; more than one embryo, if available, is transferred to the uterine cavity. This is a recognition of the low implantation rates of embryos from in vitro fertilization. A consequence of this can be high-order multiple implantation with obstetric complications. STUDY DESIGN: Retrospectively, we reviewed 42 months' in vitro fertilization experience; we related the number of embryos transferred and the pregnancy outcome. During this period 2173 fresh and frozen-thawed embryo transfers were performed. One to six embryos were transferred to women whose average age was 34.4 years (range 21 to 49). RESULTS: A total of 734 delivered pregnancies (33.8% per embryo transfer) was analyzed according to whether they were single or multiple, and this was related to the original number of embryos transferred. The overall multiple pregnancy rate was 31.3% (24.7% twins, 5.8% triplets, 0.8% quadruplets). CONCLUSIONS: There was a trend toward a higher pregnancy rate with more embryos transferred. The embryonic implantation rate, which reflects the number of embryos that implant per total transferred, was not significantly different in any one group, except in older women in whom more than one embryo was transferred. Whereas greater numbers of embryos (more than three) were transferred in couples with a poorer prognosis for successful in vitro fertilization (e.g., older women [> 36 years old], previous failure of in vitro fertilization, poor embryo quality, or severe male factor causing infertility), there still remained a significant trend toward a higher pregnancy rate when more embryos were transferred. The embryonic implantation rate did not decline in the poorer-prognosis groups (more than three embryos transferred), yet the multiple pregnancy rate was increased. Technologic procedures such as embryo biopsy for aneuploidy screening are proposed as one means to reduce embryo numbers transferred without decreasing the overall pregnancy rate.

Caprine blastocyst formation following intracytoplasmic sperm injection and defined culture.

Experiments were undertaken to develop intracytoplasmic sperm injection (ICSI) to produce caprine embryos out of the normal breeding season. Oocytes were obtained from 2-6 mm ovarian follicles at slaughter. Selected oocytes with two to four layers of cumulus cells were incubated in 1 ml of H-TCM199 supplemented with 10 micrograms each of oFSH and bLH (NHPP, NIDDK, NICHD, USDA) and 20% fetal bovine serum (FBS) in a thermos (38.5 degrees C) for 4.5 h during transportation. Then, oocytes were transferred into 75 microliters of freshly prepared maturation medium under paraffin oil and a mixture of 5% O2, 5% CO2 and 90% N2. Approximately 26 h after recovery oocytes were denuded by incubation with hyaluronidase (100 IU/ml) and pipetting and held at 38.5 degrees C for 90 min. Spermatozoa frozen in egg yolk extender were thawed in a 37 degrees C water bath for 15 s. Motile fractions were selected by swim-up, then incubated for 90 min in TALP with 10 micrograms heparin/ml. Each oocyte was positioned with its first polar body at 6 or 12 o'clock by a holding pipette. Sperm (1 microliter) were added to 10 microliters medium containing 10% polyvinylpyrrolidone. A sperm cell was aspirated into a pipette, and then injected head-first into the cytoplasm of an oocyte maintained in H-TCM199 + 20% FBS at 37 degrees C. Injected oocytes were transferred to HM and, after 90 min, cultured in 50 microliters of BSA-free synthetic oviduct fluid plus polyvinyl alcohol, citrate and non-essential amino acids. Results demonstrate that caprine blastocysts can be produced outside the breeding season by the use of frozen-thawed semen and injection of sperm cells with broken tails into ova followed by culture in defined medium.

Enhancement of outcome from intracytoplasmic sperm injection: does co-culture or assisted hatching improve implantation rates?

In two separate prospectively randomized trials, intracytoplasmic sperm injection (ICSI) cycles were studied in a controlled manner to monitor the effects of either bovine oviductal epithelial cell co-culture (n = 119) or assisted hatching by zona drilling (n = 100). In the first study, immediately following ICSI, all eggs were placed directly either onto partial monolayers of bovine oviductal cells or into regular culture medium. Although the embryo developmental rate was apparently compromised in part by the presence of the co-culture cells, ultimately there were no significant differences in either the viable pregnancy rate (31.6% co-culture versus 29.0% control) or the embryonic implantation rate (11.4% co-culture versus 13.6% control). Assisted hatching also had no significant impact on ICSI cycle outcome in terms of either the viable pregnancy rate (30.0% assisted hatching versus 32.0% control) or the embryonic implantation rate (8.5% assisted hatching versus 13.5% control). However, in female patients aged > or = 35 years, assisted hatching appeared to convey a marginally significant benefit in terms of both the viable pregnancy rate (35.5% assisted hatching versus 11.1% control) and the embryonic implantation rate (10.3% assisted hatching versus 3.1% control). It seems that the overall improvement of ICSI cycle outcome cannot be achieved by the general application of either co-culture or assisted hatching. Nevertheless, it is possible that there remain specific patient groups that might benefit from selected use of either of these modalities.

Effect of coculture on subsequent survival and implantation of cryopreserved human embryos.

PURPOSE: This retrospective analysis was designed to assess the performance of human embryos following cryopreservation based on whether they were originally developed in standard culture medium (65 cycles, 223 embryos) or cocultured on partial monolayers of bovine oviductal epithelial cells (63 cycles, 198 embryos). Embryo cryosurvival and implantation were compared between the study group and the contemporaneously match controls. RESULTS: During a 2-year period when no factors of the cryopreservation program were altered, 63 transfers of 159 surviving thawed control cleavage-stage embryos (71.3% survival) that were 54% intact gave rise to 11 viable pregnancies (17.5%/ET), to yield an implantation rate of 6.9% per embryo. Sixty-three transfers of 147 thawed cocultured embryos (74.2% survival) that were 61% intact gave an implantation rate of 13.6% per embryo that was significantly higher than the control group (P < 0.05). CONCLUSION: Coculture of embryos prior to cryopreservation does not appear to improve cryosurvival; however, it does improve implantation postthaw compared with embryos following standard culture prior to cryopreservation.

Cryopreservation of human embryos and oocytes.

Human embryo cryopreservation is now a fully established adjunct to assisted reproduction, with thawed embryos implanting at a rate approaching that of fresh embryos. As with fresh embryos, the quality of frozen-thawed embryos is affected by patient age at the time of oocyte retrieval, and by culture conditions. Ovarian stimulation incorporating gonadotropin-releasing hormone analogs does not appear to be detrimental to cryopreservation success, and natural or artificial thaw cycles seem equally effective. Successful cryopreservation seems feasible for one- to eight-cell embryos and blastocysts using a variety of protocols. Selection criteria for embryos to be frozen can vary, and embryo quality has the most significant impact on post-cryopreservation viability. Unfertilized oocyte cryopreservation is coming closer to routine clinical application.

Practical evolution and application of direct intracytoplasmic sperm injection for male factor and idiopathic fertilization failure infertilities.

OBJECTIVE: To analyze the introduction of a new assisted fertilization technique for the treatment of severe male factor and idiopathic fertilization failure infertilities. DESIGN: Retrospective analysis of 16-month clinical application of IVF-ET where insemination was performed solely by direct intracytoplasmic sperm injection. SETTING: Clinical IVF-ET program. PATIENTS: Ninety-two couples undergoing 105 cycles of sperm injection. RESULTS: One hundred embryo transfers yielded 28 viable pregnancies (28%) from which eight normal deliveries have occurred to date. Complete cleavage arrest or fertilization failure occurred in four cycles, and one couple had all embryos cryopreserved. One thousand one hundred forty-three eggs were injected of which 173 (15%) degenerated. Four hundred seventy-nine of the surviving 970 eggs became normally fertilized (49%), and 381 of these zygotes (79.5%) developed suitably for cryopreservation or for transfer. Thirty-four of 310 embryos transferred implanted, yielding an implantation rate of 11%. Both testicular and epididymal sperm were used successfully to achieve fertilization and pregnancies, as was sperm retrieved by electroejaculation. Older women and couples suffering from prior idiopathic fertilization failure had a markedly poorer outcome. CONCLUSIONS: These results confirm that the intracytoplasmic sperm injection technique is a successful form of assisted fertilization that can be applied to a wide range of couples at significant risk from fertilization failure.

Factors affecting success with intracytoplasmic sperm injection.

In this study, 141 couples underwent 163 cycles of in vitro fertilization (IVF) and embryo transfer in which the eggs were inseminated by intracytoplasmic sperm injection (ICSI). Overall, 41% of the injected eggs were normally fertilized and 81% of the resulting embryos were suitable for cryopreservation (91 embryos) or uterine transfer. From 153 fresh embryo transfers, 45 ongoing or delivered pregnancies (27.6% per cycle) were achieved, and of the 507 embryos transferred, 54 successfully implanted giving an implantation rate per embryo of 10.7%. Five additional pregnancies did not yield a viable fetus or underwent a spontaneous abortion, giving a miscarriage rate of 10% (5/50). Increased maternal age or a prior diagnosis of failed fertilization after conventional IVF had a significantly negative impact on success. Sperm from the testis and epididymis, those retrieved by electro-ejaculation, and completely immotile ejaculated sperm all gave rise to pregnancies. ICSI reinsemination was used with limited success to rescue failed fertilization cycles, although the implantation rate per embryo was poor (5%). ICSI has greatly improved the ability to use IVF for treating couples with a poor fertilization potential.

Assisted hatching with or without bovine oviductal epithelial cell co-culture for poor prognosis in-vitro fertilization patients.

Older patients and those who consistently return for embryo transfer but without implantation were studied to see if a combination of day 3 assisted hatching and co-culture (AH+CC) might be beneficial compared to assisted hatching alone (AH-alone). Female patients of > or = 38 years and couples who had previously failed to implant embryos three times or more were prospectively and randomly assigned to either an experimental or a control group. In the experimental group all embryos were co-cultured on partial monolayers of bovine oviductal epithelial cells for 2 days followed by assisted hatching by zona drilling (AH+CC). All control embryos were cultured by standard procedures until day 3 when they also underwent zona drilling prior to uterine transfer (AH-alone). With 50 cycles in each group there was unfortunately a marginal bias against the AH+CC group in that these patients had undergone a higher number of previous transfer cycles. There was a marginally lower percentage of fragmentation and a significantly higher degree of zona thickness variability in the AH+CC embryo group. Embryonic implantation was significantly increased (P < 0.05) in the AH+CC group (18%) when compared to the AH-alone group (10%). This difference was reflected in a significantly higher (P < 0.05) initial pregnancy rate (52 versus 32%) in the AH+CC group, and a higher (not significant) viable pregnancy rate (38 versus 22%).

Treatment of male infertility and idiopathic failure to fertilize in vitro with under zona insemination and direct egg injection.

OBJECTIVE: Failure to fertilize eggs in vitro may be countered by micromanipulation of gametes to place selected spermatozoa underneath the zona pellucida of the egg or directly into the egg, thereby improving chances of fertilization and production of viable embryos. Analysis of our clinical data for assisted fertilization was undertaken to assess those factors of relevance in this therapy, and a description of our procedures are given. STUDY DESIGN: Retrospective analysis of 85 cycles (73 couples) of in vitro fertilization and embryo transfer performed at a private infertility clinic, in which micromanipulation for assisted fertilization was used to overcome either severe male factor infertility or idiopathic failure to fertilize, was performed. RESULTS: In 60 cycles where only embryos from under zona insemination were available for uterine transfer, 15 singleton and two twin pregnancies occurred (28.3% viable pregnancy rate per transfer, 14.1% embryonic implantation). In 14 of these cycles embryos arose only after repeated under zona insemination adding more spermatozoa; this accounted for four of the singleton and one of the twin pregnancies (38.5% pregnancy rate, 22.2% embryonic implantation). No embryos arose from partial zona dissection performed in five cycles on sibling eggs. However, in 16 cycles conventional insemination yielded fertilization in six cycles, and mixed transfer of these embryos and sibling embryos from under zona insemination gave rise to one pregnancy from four transfers (pregnancy rate 25%, embryonic implantation 7.1%). Likewise, in nine cycles donor spermatozoa yielded fertilization in eight cycles, and mixed transfer with sibling embryos fertilized by under zona insemination with partner's spermatozoa gave rise to two pregnancies from five transfers (pregnancy rate 40%, embryonic implantation 15.8%). Fertilization and pregnancy rates did not differ whether couples suffered either from male factor infertility or from previous idiopathic fertilization failure. Direct egg injection of a single spermatozoon into 105 eggs gave an 88.6% egg survival and 32.3% fertilization. Mixed transfers with sibling embryos from conventional and under zona insemination yielded one triplet, one twin, and three singleton pregnancies. CONCLUSIONS: Overall, a 24.7% (21/85) viable pregnancy rate per cycle initiated occurred when only embryos from assisted fertilization were available. This strongly indicates that assisted fertilization made a real contribution in cases where either insufficient spermatozoa were available for conventional insemination or in cases where previous fertilization failure had arisen. The wide range of seminal parameters were found to be unhelpful in defining chances of success with assisted fertilization.

Leuprolide acetate: serum and follicular fluid concentrations and effects on human fertilization, embryo growth, and granulosa-lutein cell progesterone accumulation in vitro.

Data from various animal models have demonstrated significant extrapituitary effects of gonadotropin-releasing hormone agonists. The purpose of this study was to determine the effects of therapeutic concentrations of leuprolide acetate on human granulosa-lutein cell steroidogenesis, fertilization, and embryo growth rates in vitro. During leuprolide administration, mean serum concentrations of leuprolide were less than 50 ng/ml and were undetectable 48 hours after cessation of administration. There was no effect of leuprolide on progesterone (P) secretion by granulosa-lutein cells cultured in the presence or absence of human chorionic gonadotropin. The effect of leuprolide on embryo growth rates was evaluated with the mouse two-cell embryo culture model and a retrospective review of early embryo growth rates in humans receiving adjunctive leuprolide therapy. There was no measurable effect of leuprolide on early embryo growth in either species. These data indicate that (1) serum and follicular and peritoneal fluid concentrations are undetectable 2 days after discontinuation of leuprolide; (2) there is no measurable effect of leuprolide on human or murine embryo growth rates up to the 8 cell stage in vitro; and (3) there is no measurable effect of leuprolide on granulosa-lutein cell P accumulation.

Percutaneous nephrolithotomy. The first 50 cases.

Initial experience at two centres in Cape Town with a new technique for the removal of urinary calculi is described. The technical difficulties encountered and the methods used to overcome them are outlined. Percutaneous nephrolithotomy has rendered conventional open surgery largely obsolete for treatment of stone disease of the upper urinary tract.

Computed tomographic and ultrasonic localization of adrenal mass lesions.

A study of 20 patients with adrenal mass lesions shown on computed tomography (CT) is reported. CT accurately localized a unilateral adrenal mass in 12 patients with Cushing's syndrome, primary hyperaldosteronism and phaeochromocytoma. Unsuspected primary or metastatic neoplasms of the adrenal glands were detected in 8 patients who underwent CT for other reasons. In 9 cases ultrasonography was also useful in localizing large adrenal masses. This study confirms the safety and accuracy of CT and ultrasonography in evaluating patients with adrenal masses, thereby obviating the need for more invasive diagnostic procedures.

Air in unusual places: some causes and ramifications of pneumomediastinum.

Five unusual cases of pneumomediastinum are described. In three the probable cause was thoraco-abdominal straining against a closed glottis during violent exercise, in criminal assault, or competitive sport. The resultant increase in intra-alveolar pressure produces an air leak which passes via the pulmonary interstitium into the mediastinum. It can then pass up into the neck to produce widespread subcutaneous emphysema and down through the diaphragmatic hiatuses to produce extraperitoneal emphysema. This may outline the lower surface of the diaphragm to stimulate intraperitoneal air, but it can also leak through the parietal peritoneum to result in actual intraperitoneal air. Therefore, in the patient who has been hospitalised after violent physical stress with or without blunt trauma, pneumomediastinum does not necessarily indicate tracheobronchial or oesophageal rupture and subdiaphragmatic air does not necessarily indicate bowel rupture. Probably any form of exercise in which the Valsalva manoeuvre is performed may cause pneumomediastinum, as may other causes of increased intra-alveolar pressure such as mechanical ventilation, bronchospasm, coughing and vomiting. Vomiting is a likely contributing cause in the pneumomediastinum of diabetic ketosis, of which a case is described. Another case is presented in which air passed in the opposite direction, from perforated extraperitoneal bowel up into the mediastinum.