Propofol concentrations in follicular fluid during general anaesthesia for transvaginal oocyte retrieval.

Propofol (Diprivan) is an i.v. anaesthetic used for general anaesthesia. The purpose of this study was to measure the propofol concentration in arterial blood and follicular fluid in patients during transvaginal oocyte retrieval. After approval by the University Ethics Committee, 30 women participated in this prospective study. Following induction of anaesthesia with 0.5 mg alfentanil and 2 mg.kg-1 propofol i.v., a continuous infusion of propofol at 10 mg.kg-1.h-1 was used for maintenance of anaesthesia. Follicular fluid and arterial blood samples were aspirated simultaneously at fixed intervals during the surgical procedure and propofol assayed by high pressure liquid chromatography (HPLC). The mean follicular fluid concentration of propofol increased linearly with time from 0.10 +/- 0.02 microgram.ml-1 to 0.57 +/- 0.06 microgram.ml-1 and was strongly related to the cumulative dose of propofol administered. The absorption of propofol was time-dependent. There was no correlation between the concentration of propofol in the follicular fluid and the arterial blood concentration of the drug. In conclusion, a propofol-based anaesthetic technique resulted in significant concentrations of this agent in follicular fluid, related to the dose administered and to the duration of propofol administration.

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.

Controlled comparison of commercial media for human in-vitro fertilization: Ménézo B2 medium versus Medi-Cult universal and BM1 medium.

For many years, we have been using Ménézo B2 medium in our human in-vitro fertilization (IVF) programme. Ménézo B2 contains bovine serum albumin, which has in the meantime become less popular. The search for a valid alternative has therefore been initiated. In a first study from December 1993 to June 1994, we compared Ménézo B2 medium to Medi-Cult (Medi-Cult Universal IVF medium). In this auto-controlled study, sibling oocytes cultured in Ménézo B2 showed a normal fertilization rate similar to that for those cultured in Medi-Cult: 62.9 +/- 33.3 and 61.0 +/- 33.0% respectively (NS; paired t-test). In a second auto-controlled study conducted from November 1995 to March 1996, we compared Ménézo B2 to BM1 (Ellios Bio-Media) medium. The normal fertilization rate in the sibling oocytes was similar for those cultured in Ménézo B2 and in BM1: 66.9 +/- 27.5 versus 62.0 +/- 31.7% (NS; paired t-test). After further culture of the fertilized oocytes, no difference was apparent in either study as regards the morphological characteristics of the embryos cultured in the different media. The speed of development, however, did differ. In both studies, we observed a higher proportion of the embryos reaching at least the three- to four-cell stage at approximately 41 h post-insemination in Ménézo B2 medium than in either Medi-Cult or BM1. As regards the implantation potency of the embryos obtained in the different media, data were collected from the non-randomized transfers. These data indicate no difference between the media tested. This study indicates that the three commercial media with a different composition are equally able to sustain fertilization and embryo development until transfer.

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.

Results of intracytoplasmic sperm injection with ejaculated, fresh and frozen-thawed epididymal and testicular spermatozoa.

Over the past 5 years, intracytoplasmic sperm injection (ICSI) has been developed to alleviate severe long-standing andrological infertility in couples who could not be helped by standard in-vitro fertilization (IVF), or who could not undergo IVF because too few motile and morphologically normal spermatozoa were present in the ejaculate. ICSI can also be carried out with fresh and frozen-thawed epididymal spermatozoa from patients with obstructive azoospermia and with testicular spermatozoa from most patients with obstructive azoospermia and from some patients with non-obstructive azoospermia. This review reports the results of 5 years of ICSI practice (1991-1995) at the Brussels Free University Centre for Reproductive Medicine, including selection of patients, oocyte and spermatozoa for ICSI, ICSI procedures and its assessment. ICSI was performed in 97% of the 4688 planned treatment cycles. Ejaculated spermatozoa were used in 88% of the cycles, epididymal spermatozoa in 5% of the cycles and testicular spermatozoa in 7% of the cycles. ICSI was performed on 48393 metaphase II oocytes. Of these, 89.5% were morphologically intact, and 70.9% of the intact oocytes were normally fertilized; 64.9% of the two-pronuclear oocytes developed after a further 24 h of in-vitro culture into cleaved embryos, which were transferred or cryopreserved for subsequent use. After transfer of fresh embryos, the percentage of cycles with positive serum human chorionic gonadotrophin was 36.2% per transfer and the percentage of deliveries was 27.0% per transfer. The performance of ICSI with regard to damage, pronuclear status, embryo development and transfer was analysed separately for the four different types of spermatozoa used: fresh ejaculated, epididymal and testicular as well as frozen-thawed epididymal spermatozoa.

Microsurgical epididymal sperm aspiration with motile trophozoite cells but no spermatozoa.

This paper reports on a patient in whom the clinical diagnosis of obstructive azoospermia was made according to clinical observations, i.e. azoospermia, normal andrological examination, normal follicle stimulating hormone and a misleading histopathological report of a testicular biopsy. Microsurgical vasoepididymostomy failed to restore fertility, and as a last resort, microsurgical sperm aspiration was performed. Although flagellated cells were observed in the epididymal aspiration, no spermatozoa were observed and wet preparation of multiple testicular biopsies failed to demonstrate any spermatozoon. This patient was diagnosed to have a non-obstructive azoospermia, resulting from maturation arrest associated with trichomonas infection at the level of the epididymis.

The effect of propofol on parthenogenetic activation, in vitro fertilization and early development of mouse oocytes.

OBJECTIVE: To assess the effect of propofol on fertilization and early embryo development in a mouse IVF model. DESIGN: Controlled study. SETTING: Mouse IVF. INTERVENTION(S): Mouse oocytes were exposed in vitro to propofol at a concentration of 0 (control), 50, 250, 500, 1,000, or 5,000 ng/mL for 30 minutes, washed, and inseminated. Thereafter, fertilization was assessed. Subsequent in vitro development to the blastocyst stage was monitored daily. The potential to activate parthenogenetically oocytes also was evaluated by looking for spontaneous extrusion of the second polar body or development to the two-cell stage. In a second step, a pure propofol solution was added to culture medium and used as a standard. MAIN OUTCOME MEASURE(S): Two-cell and blastocyst-stage embryo. RESULT(S): Where fertilization occurred, subsequent embryo cleavage and development up to the blastocyst stage was affected significantly by the presence of propofol solution in the medium, (i.e., 3% to 41%) in comparison with the control group (76%). Exposure of unfertilized oocytes for 30 minutes to propofol results in a parthenogenetic activation of 33% to 60%, which was significantly higher than the control (10%). When oocytes were kept in propofol for 24 hours, a mean of 30% of activation was observed as compared with 0.5% for the control. CONCLUSION(S): We can conclude from these experiments that even a brief exposure of cumulus-enclosed oocytes to a low concentration of propofol is deleterious to subsequent cleavage. Exposure of unfertilized oocytes to propofol results in a high degree of parthenogenetic activation.

Review: preimplantation diagnosis of inherited disease.

Preimplantation diagnosis of inherited diseases has become possible with the techniques of in vitro fertilization, blastomere biopsy of the 6- to 10-cell embryo and DNA analysis of the single blastomeres. Disease-free embryos are selected for transfer to the uterus, thereby avoiding the need for termination of a fetus diagnosed as affected in prenatal diagnosis in the first or early-second trimester of pregnancy. The genetic indications for preimplantation diagnosis are theoretically the same as for prenatal diagnosis, but the defects must be detectable by the polymerase chain reaction. For X-linked recessive diseases, fluorescence in situ hybridization can be used as an alternative for the selection of female embryos. So far almost 40 healthy children have been born worldwide after preimplantation diagnosis for genetic disease. The possibilities and limitations of preimplantation diagnosis, especially in prevention of inherited disease, are discussed in this review.

One year's experience with elective transfer of two good quality embryos in the human in-vitro fertilization and intracytoplasmic sperm injection programmes.

High incidences of multiple pregnancies, after transferring a maximum of three embryos, were observed after in-vitro fertilization (IVF) treatment. In a randomized study, it was demonstrated that, after taking into account embryo quality and other positively interfering parameters, an elective transfer of two good quality embryos does not significantly influence the pregnancy rate. The intracytoplasmic sperm injection (ICSI) technique was successfully developed in the meantime and high incidences of multiple pregnancies were also obtained after ICSI. The question arose whether after ICSI there was also room for elective double embryo transfer in a well-defined patient group. This report covers 1 year of IVF and ICSI treatment and the results are presented in relation to the number of embryos transferred. The embryo development is similar for zygotes obtained after IVF and ICSI; for both techniques 63% of the zygotes develop to type A-B embryos and 13% to type C embryos. There is also no difference in the pregnancy rate after ICSI or IVF. Globally, after IVF, 307 out of the 766 double and triple transfers (40.1%) and 317 out of 774 double and triple transfers (40.9%) after ICSI resulted in a positive HCG. After IVF, 73.9% (227) and after ICSI 76.3% (242) of the pregnancies were evolutive. Neither was there any difference between the two techniques as regards the implantation rate per transferred embryo. After IVF, 22.8% of the transferred embryos implanted compared with 21.8% after ICSI. When the elective double embryo transfers were compared, no difference was found between IVF and ICSI. After IVF, 102 of the 211 elective double transfers (48.1%) resulted in a pregnancy versus 93 out of 225 (41.3%) after ICSI [not significant (NS)]. A high implantation rate per transferred embryo (IVF: 33.2%; ICSI: 26.9%, NS) was obtained in this elective double transfer category, as was also reported in the randomized study. These data confirm the results obtained in our randomized study and the effectiveness of the elective double embryo transfer for IVF as well as for ICSI.

Results of 55 intracytoplasmic sperm injection cycles in the treatment of male-immunological infertility.

Antisperm antibodies present in the semen can be a primary cause of infertility. If the proportion of spermatozoa carrying antisperm antibodies is very high, then usually a poor result ensues in standard in-vitro fertilization. We therefore employed intracytoplasmic sperm injection (ICSI) in 55 cycles (37 patients) where the proportion of antisperm antibody-bound spermatozoa was 80% or higher, as determined by the mixed antiglobulin reaction (MAR) test. The type and location of antisperm antibodies were determined by the immunobead test in 30 of the 37 patients. The mean normal fertilization rate was 75.7% in these 55 cycles, which was significantly higher than the fertilization rate in another 1767 ICSI cycles (69.2%) performed over the same period and where MAR-negative semen (the level of antisperm antibodies was < 80%) was used for microinjection. Embryonic development was comparable, but a higher proportion of poor-quality embryos was obtained with MAR-positive than with MAR-negative semen samples. Out of the 55 patients, 53 had embryos replaced (96.4%) and a fetal sac was detected by ultrasonography in 14 patients (26.4%). The data indicate that fertilization, embryo development and pregnancy rates after ICSI are not influenced significantly by the proportion of antisperm antibody-bound spermatozoa, nor by the dominant type of antibodies present, nor by the location of the antisperm antibody on the spermatozoa. The conclusion of this study is that ICSI should be the primary choice for patients who have high numbers of antisperm antibodies present in their semen.

Maturation of human cumulus-free germinal vesicle-stage oocytes to metaphase II by coculture with monolayer Vero cells.

The objective of this study was to evaluate and compare the efficacy of coculture with Vero cells (monkey kidney cells) and in-vitro culture in B2 Ménézo medium for the maturation of germinal vesicle (GV)-stage cumulus-free human oocytes to metaphase II. Cumulus-free human GV-stage oocytes obtained after ovulation induction and retrieval via vaginal ultrasound echo-guided puncture were put either in coculture with Vero cells or in 25 microliters droplets of B2 Ménézo medium. In a controlled randomized study, 145 GV-stage oocytes were matured in vitro: 72 were placed in the coculture system with Vero cells and 73 were cultured in B2 alone. After 30 h of in-vitro culture, 82.4% had dissolved the nuclear membrane and extruded the first polar body in the coculture system compared with 37% in B2 medium (P < 0.001). A significant difference was also noticed in the number of GV-stage oocytes which underwent GV breakdown; the proportion of oocytes which did not extrude the first polar body (metaphase I) was 1.6% in the coculture system compared to 44.0% in the B2 medium. It can be concluded that coculture with Vero cells improves the in-vitro maturation of cumulus-free human GV-stage oocytes.

The effect of pentoxifylline on in-vitro fertilization in the presence of anti-sperm antibodies.

In cases of severe immunological male-factor infertility, impairment of spermatozoal motility and of acrosome reaction resulting in reduced fertilization capacity have been described by several authors. The present study investigated the use of pentoxifylline in enhancing in-vitro fertilization (IVF) in the presence of anti-sperm antibodies. Thirty-seven IVF cycles were conducted in 28 different couples suffering from immunological male-factor infertility with at least 50% antibody-coated spermatozoa. Sibling oocytes were inseminated at random with spermatozoa incubated with or without 3.6 mM pentoxifylline after selection by a Percoll gradient. No difference in motility of the final sperm preparations was observed prior to insemination. Fertilization rate, cleavage rate and embryo quality were similar in both treatment and control groups. Nine out of ten pregnancies were achieved after the replacement of embryos both from the treatment and control group. Although pentoxifylline is known to enhance motility in-vitro and to promote induced acrosomal loss, its indiscriminate use failed to improve IVF performance in patients with anti-sperm antibodies. Further research may be necessary in order to elucidate whether a given subpopulation of these patients may benefit from a selective application of pentoxifylline.

Controlled comparison of Earle's balanced salt solution with Ménézo B2 medium for human in-vitro fertilization performance.

In order to optimize the in-vitro fertilization (IVF) procedure and to avoid the batch-to-batch fluctuations of in-house culture medium, we investigated the use of a commercially available medium. In our human IVF programme, a controlled comparison was performed from July 1991 to February 1992 between in-house Earle's medium and commercially available Ménézo B2 medium. The major difference between the two tested media is the additional presence of amino acids and water-soluble vitamins in Ménézo B2 and the different origins of the proteins source, i.e. bovine serum albumin (BSA) in Ménézo B2 and human serum albumin (HSA) in Earle's medium. In the first, auto-controlled study, sibling oocytes cultured in Ménézo B2 showed a significantly higher fertilization rate than those cultured in Earle's medium: 58.1 +/- 37.2% and 52.1 +/- 36.9% respectively. After further culture of the fertilized oocytes, there was no difference in the morphological characteristics of the embryos cultured in the different media, although the rate of development was different. In Ménézo B2 medium, a higher proportion of the embryos had reached at least the 4-cell stage at transfer. In the second, randomized study, effects on the pregnancy and implantation rates of both media were analysed. In the group of oocytes cultured in B2, more excellent or good-quality embryos were available for transfer at a more advanced stage of development. No significant difference in pregnancy rate was observed: 39.1% per transfer in Earle's medium versus 40.5% per transfer in Ménézo B2.(ABSTRACT TRUNCATED AT 250 WORDS)

Avoidance of triplet pregnancies by elective transfer of two good quality embryos.

Attempts to increase the probability of a successful pregnancy in in-vitro fertilization (IVF) treatment by increasing the number of embryos transferred automatically also increase the probability of multiple pregnancies and their attendant risks. Even where the number of transferred embryos is limited to a maximum of three as in this and other centres, there is a high incidence of twins and triplets. The question therefore arises whether the number of transferred embryos should be further limited to a maximum of two in cases where the prognosis is otherwise good. The only objection to this idea is a possible lowering of pregnancy rate. The present study set out to investigate this question. No significant lowering of pregnancy rate was found, so that limiting the number of transferred embryos to two where the prognosis is otherwise good has now become standard practice in our centre. A good IVF prognosis was defined by the following criteria: first attempt for IVF, less than 37 years old, and good embryo development. From 183 patients fulfilling these criteria, 80 agreed to the transfer of two embryos (group 1) and 103 opted for a triple transfer (group 2). Patient characteristics and embryology results were similar in the two groups. In group 1, 34 patients (42.5%) became pregnant and in group 2, 50 (48.5%). This difference is not significant. Similarly, twin pregnancy rates in both groups were high; eight twin pregnancies (23.5%) in group 1 and 12 (24%) in group 2.(ABSTRACT TRUNCATED AT 250 WORDS)

Higher success rate by intracytoplasmic sperm injection than by subzonal insemination. Report of a second series of 300 consecutive treatment cycles.

Subzonal insemination (SUZI) and intracytoplasmic sperm injection (ICSI) were carried out in 300 treatment cycles in couples unable to be helped by conventional in-vitro fertilization treatment. More oocytes were damaged by ICSI (13.5%) than by SUZI (7.1%). The normal fertilization rate was substantially higher after ICSI (51.0%) than after SUZI (14.3%) and was related to the semen characteristics. The cleavage rate was similar for both procedures (77%). After 217 embryo transfers (72.3% of the treatment cycles) 66 pregnancies were established, i.e. pregnancy rates of 22.0% per started cycle and 30.4% per embryo transfer. So far, pregnancy loss has occurred in 27.3% of the pregnancies, nine healthy children have been born after eight deliveries and 41 clinical pregnancies are progressing uneventfully. Chorionic villus sampling or amniocentesis have been performed in 35 pregnancies and 39 normal fetal karyotypes have been obtained after cytogenetic analysis.

Cytogenetic and morphological observations of single pronucleated human oocytes after in-vitro fertilization.

At 16-18 h after insemination, 5.5% of the inseminated oocytes displayed only one pronucleus. The incidence of single-pronucleated oocytes was not correlated with the age of the patient. Cytogenetic analysis of 41 embryos derived from single-pronucleated oocytes revealed a haploid chromosome complement in 12.2%, a diploid chromosome complement in 80.5% and a triploid set in 7.3% of the embryos. In one diploid metaphase, a Y chromosome could be clearly demonstrated. A total of 312 single-pronucleated oocytes were evaluated twice (at 16-18 h and at 20-24 h after insemination). In 25% of the single-pronucleated oocytes a second pronucleus was observed 4-6 h later, suggesting asynchronous or delayed pronuclear formation. The cleavage of these embryos was similar to the cleavage of embryos with two pronuclei at the first evaluation. Parthenogenetic activation and asynchronous pronuclei may both be mechanisms leading to the morphological observation of a single pronucleus. In clinical practice, the second repeat observation of single-pronucleated oocytes became part of the standard procedure.