Pregnancies after in vitro fertilization and transfer of human blastocysts.

In a study of 29 cycles of IVF, ET was performed on day 5 after oocyte recovery when embryos had developed to the morula/blastocyst stage. Three preclinical pregnancies and three live births resulted (2 singleton and 1 twin), giving a viable PR per ET of 10%. It is concluded that while day 5 ET may well be important in terms of embryo biopsy for the preimplantation diagnosis of genetic disease, day 2 ET remains preferable for therapeutic IVF. Although these data would not support the introduction of day 5 ET into routine therapeutic IVF, delayed ET should be considered as an alternative approach to preimplantation diagnosis. Indeed, because the latter will generally involve the treatment of normal, fertile couples, it might be predicted that embryo survival rates, and thus the rate of pregnancy after day 5 ET, would be better than those presented here.

Pregnancies following in vitro fertilization and ultrasound-directed surgical embryo transfer by perurethral and transvaginal techniques.

Ultrasound-directed surgical ET is useful in patients with a history of difficult cervical (nonsurgical) transfer, and was performed without complications in the small group reported here. The technique is straightforward, and requires no greater expertise than that necessary for ultrasound-guided oocyte collection. However, further studies are necessary to assess its role in the routine transfer of embryos following IVF.

Removal of bacterial contaminants from semen for in vitro fertilization or artificial insemination by the use of buoyant density centrifugation.

Buoyant density centrifugation of semen produces the accumulation of populations of highly motile, morphologically normal spermatozoa in the lowermost 1 ml of Percoll (Pharmacia Fine Chemicals AB, Uppsala, Sweden) density gradients. In addition, the majority of bacteria present in semen are retained in the seminal plasma at the top of the gradients. Of 40 semen samples examined, 37 contained detectable bacteria, but after buoyant density centrifugation, the spermatozoal populations collected from the lowermost 1 ml of the Percoll columns were found to contain few or no bacteria. When preparations were collected using sterile technique (by boring a hole through the bottom of the centrifuge tube), 14 of the 20 preparations were found to be bacteria-free. When preparations were collected by passing a spinal needle from the surface through the seminal plasma to the bottom of the centrifuge tube, the sterility of the final spermatozoa preparations was not maintained, with only 5 of the 20 samples completely free of bacteria. The residual bacterial contamination of the remaining 15 samples was, however, very low (less than 5 colonies after a 48-hour culture period).

Laparoscopic zygote intrafallopian transfer using augmented local anesthesia.

In this study, 29 laparoscopic ZIFTs were performed in 21 patients using local anesthesia augmented with intravenous analgesia. The technique was well tolerated; significant discomfort arose only when the fallopian tubes were manipulated and was minimized by transferring zygotes to one tube only. Seven pregnancies resulted, of which three have delivered and one is ongoing.

Are human spermatozoa separated on a Percoll density gradient safe for therapeutic use?

Motile morphologically normal human spermatozoa can be separated from semen by buoyant density centrifugation on Percoll (Pharmacia Fine Chemicals AB, Uppsala, Sweden) gradients. In this study, the authors have examined (1) the efficiency of washing procedures to remove contaminating Percoll particles from the separated spermatozoa, and (2) the potential of Percoll particles, which contain silica, to cause an inflammatory response when used for intrauterine insemination, or when introduced into the fallopian tube during gamete intrafallopian transfer (GIFT) procedures, as assessed by an intraperitoneal injection into mice. Although Percoll was phagocytosed at the injection site, and therefore cannot be presumed to be totally inert, no generalized inflammatory response was detected. A double spin and wash technique was found to remove most residual Percoll from the spermatozoa, as assessed by scanning electron microscopy. These results suggest that procedures involving the use of Percoll for the separation of human spermatozoa for in vitro fertilization, GIFT, or intrauterine insemination should include stringent washing protocols that will remove most, if not all, contaminating Percoll from the sample.

The relationship between chromosomal abnormality in the human preimplantation embryo and development in vitro.

The relationship between the survival of the human preimplantation embryo in vitro and chromosomal abnormality was investigated by cytogenetic analysis of a total of 250 embryos of varying morphology between the pronucleate stage and the 8-cell stage. The overall incidence of chromosomal abnormality among these embryos was 49%. At the pronucleate stage (n = 46) the incidence was 65.2%, at the 2-4-cell stage (n = 126) it was 54.6%, and at the 5-8-cell stage (n = 78) it was 27.4%. Cleavage-stage embryos with poor morphology (irregular shaped blastomeres with severe extracellular fragmentation) showed a higher incidence of chromosomal abnormality (62%; 54 of 87 analysed) than those with good morphology (22.2%; 26 of 117 analysed). This study demonstrates: (i) that there is progressive loss of chromosomally-abnormal embryos during preimplantation development; and (ii) that there is an association between chromosomal abnormality and embryo morphology.

Cytogenetic analysis of human preimplantation embryos following developmental arrest in vitro.

The relationship between chromosomal abnormalities in the human preimplantation embryo and developmental arrest in vitro was investigated. Cytogenetic analysis of 171 embryos that had arrested between the pronucleate and the 8-cell stages demonstrated that the overall incidence of chromosomal abnormality among these embryos was 63.4%. Of the embryos that arrested at the pronucleate stage (n = 48), 47.9% were chromosomally abnormal, compared with 59.5% of those that arrested between the 2- and 4-cell stages (n = 50), and 82.8% of those arrested between the 5- and 8-cell stage (n = 73). The rate of abnormality in embryos with poor morphology (irregular shaped blastomeres and considerable extracellular fragmentation) was significantly higher (86.8%; n = 33) than those with good morphology (60%; n = 51; P<0.005). These results suggest that there is an association between chromosomal abnormality, developmental arrest in vitro, and poor morphology.

Superovulation, IGFBP-1 and birth weight.

In this study, the effect of superovulation on the circulating levels of insulin-like growth factor binding protein-1 (IGFBP-1) has been investigated. IGFBP-1 levels were measured in singleton pregnancies achieved either naturally (n = 203) or following superovulation, in-vitro fertilisation and embryo transfer (IVF-ET) with either pituitary desensitisation with buserelin and superovulation with human menopausal gonadotrophin (b/hMG) followed by IVF-ET (n = 15) or with clomiphene citrate and hMG (CC/hMG) followed by IVF-ET (n = 15, 1st trimester only). The circulating levels of IGFBP-1 were similar in all three groups during the first trimester, and in both normal and b/hMG pregnancies in the second, but were significantly higher during the third trimester in b/hMG pregnancies than in normal pregnancies (P = 0.0002). The birth weights were significantly lower in the b/hMG group (P = 0.04), but not in the CC/hMG group compared with natural conceptions. Gestational age at delivery was similar in control and b/hMG pregnancies, but significantly reduced in CC/hMG pregnancies (P = 0.04). These data suggest that pregnancies achieved following superovulation with b/hMG are associated with elevated levels of IGFBP-1 during the third trimester of pregnancy and reduced birth weight.

Live births after intracytoplasmic sperm injection in the management of oligospermia and azoospermia in Nigeria.

Intracytoplasmic sperm injection has revolutionised the management of male infertility. We report two cases that demonstrate the successful application of this technology in Nigeria in the management of both oligospermia and azoospermia. The first case relates to the treatment of a 31-year-old woman who required intracytoplasmic sperm injection of her husband's sperm for the treatment of both tubal fertility and male infertility. She had three embryos transferred on 9th June 1999 and was delivered of healthy male and female infants by caesarean section in January 2000 at 33 weeks gestation. The second case describes a 38-year-old woman who required intracytoplasmic sperm injection of the husband's surgically collected sperm for the management of azoospermia. She had two embryos transferred on 16th December 1999 and was delivered of a healthy male infant by caesarean section on 19th July 2001.

GSK962040: a small molecule, selective motilin receptor agonist, effective as a stimulant of human and rabbit gastrointestinal motility.

There is an urgent clinical need for a safe, efficacious stimulant of gastric emptying; current therapies include erythromycin (an antibiotic with additional properties which preclude chronic use) and metoclopramide (a 5-hydroxytryptamine type 4 receptor agonist and an antagonist at brain D2 receptors, associated with movement disorders). To move away from the complex motilide structure of erythromycin, a small molecule motilin receptor agonist, GSK962040, was identified and characterized. The compound was evaluated using recombinant human receptors, rabbit and human isolated stomach preparations known to respond to motilin and in vivo, by measuring its ability to increase defecation in conscious rabbits. At the human motilin receptor, the pEC50 (the negative logarithm to base 10 of the EC50 value, the concentration of agonist that produces 50% of the maximal response) values for GSK962040 and erythromycin as agonists were, respectively, 7.9 and 7.3; GSK962040 had no significant activity at a range of other receptors (including ghrelin), ion channels and enzymes. In rabbit gastric antrum, GSK962040 300 nmol L(-1)-10 micromol L(-1) caused a prolonged facilitation of the amplitude of cholinergically mediated contractions, to a maximum of 248 +/- 47% at 3 micromol L(-1). In human-isolated stomach, GSK962040 10 micromol L(-1), erythromycin 10 micromol L(-1) and [Nle13]-motilin 100 nmol L(-1), each caused muscle contraction of similar amplitude. In conscious rabbits, intravenous doses of 5 mg kg(-1) GSK962040 or 10 mg kg(-1) erythromycin significantly increased faecal output over a 2-h period. Together, these data show that GSK962040, a non-motilide structure, selectively activates the motilin receptor. Simplification of the structural requirements to activate this receptor greatly facilitates the design of potentially new medicines for gastroparesis.

Preparation of human spermatozoa for in vitro fertilization by isopycnic centrifugation on self-generating density gradients.

A simple and rapid method is described for the retrieval of highly motile, morphologically normal spermatozoa from human semen. This method is a modification of a technique described previously and employs the process of isopycnic centrifugation of semen on self-generating Percoll density gradients. The procedure is carried out under sterile conditions and has no detectable deleterious effects on the fertilizing capacity of spermatozoa. Spermatozoa may be recovered from semen samples of widely differing quality and can be used successfully for in vitro fertilization (IVF). This technique may be useful not only for the preparation of spermatozoa for IVF and possibly for artificial insemination by husband (AIH) but also for the investigation and analysis of the causes of infertility associated with oligozoospermia.

Immaturity and chromosomal abnormalities in oocytes that fail to develop pronuclei following insemination in vitro.

A total of 293 oocytes that failed to develop pronuclei after insemination in vitro with apparently normal, fertile spermatozoa were obtained from 87 women undergoing therapeutic in-vitro fertilization. The oocytes were investigated cytogenetically to determine the incidence of immaturity and chromosomal abnormalities. Cytogenetic examination was possible in 81% of the preparations, in which immaturity and chromosomal abnormalities were present in 29.5 and 58.7% respectively.

Polypeptide profiles of human oocytes and preimplantation embryos.

The polypeptides that direct fertilization and early development until activation of the embryonic genome occurs, at the 4-8 cell stage in the human, are exclusively maternal in origin, and are either synthesized during oogenesis or translated later from maternal mRNA. Using sodium dodecyl sulphate-polyacrylamide gel electrophoresis and silver stain, we have visualized and compared the polypeptides present in different populations of human oocytes and cleavage stage embryos obtained after superovulation and insemination in vitro. Two polypeptide patterns were resolved, differing in the region of mol. wt 69 kDa. The distribution of these patterns showed no correlation with the ability of individual oocytes to achieve fertilization and develop normally to the 8-cell stage.

The relationship between chromosomal abnormalities in the human oocyte and fertilization in vitro.

The relationship between chromosomal abnormalities in the human oocyte and fertilization in vitro was investigated by cytogenetic analysis of an unselected population of oocytes, where failure to achieve fertilization was attributed to dysfunctional spermatozoa. The results demonstrated that 47% of such oocytes were chromosomally abnormal. These data were used to calculate that the incidence of chromosomal abnormalities in oocytes that do, and those that do not develop pronuclei following insemination in vitro is 26.6% and 20.4% respectively. Statistical analysis demonstrated no relationship between chromosomal abnormality in the oocyte and its capacity to achieve fertilization in vitro.

Sequence and regulation of morphological and molecular events during the first cell cycle of mouse embryogenesis.

Mouse oocytes were fertilized in vitro and the precise timing and sequence of morphological and molecular events occurring during the first cell cycle were investigated. The timing of development through the first cell cycle was found to be initiated by an event associated with sperm penetration rather than with germinal vesicle breakdown. DNA replication is initiated randomly in either pronucleus of a given egg, beginning approximately 11 h post insemination (hpi), and S phase lasting 6-7 h in both. Careful study of polypeptide synthetic profiles revealed three classes of changes in polypeptide synthesis during the first few hours of development: fertilization-independent, fertilization-accelerated, and fertilization-dependent. Pulse-chase experiments and in vitro translation of extracted mRNA showed that the changes in polypeptide synthetic profile result from differential mRNA activation, differential polypeptide turnover and post-translational modifications. These results support the notion that following ovulation, development is controlled at two levels. An endogenous (oocyte) programme, set in train by the terminal events of oocyte maturation, may regulate the 'housekeeping' functions of the egg, while sperm penetration activates a further endogenous (fertilization) programme, which may serve to initiate subsequent embryogenesis.

A prospective comparison of 'in house' and commercially prepared Earle's balanced salt solution in human in-vitro fertilization.

A prospective, randomized study was undertaken to compare the use of Earle's balanced salt solution (EBSS) prepared 'in house' with that produced commercially, in 448 cycles of therapeutic in-vitro fertilization. Outcome was assessed in terms of fertilization and cleavage rates, embryo morphology, and implantation rates following embryo transfer. The only differences that were found between the two media in any of the outcome parameters were in the number of cycles with failed fertilization (1/218 in 'in house' medium compared with 10/230 in commercially prepared medium; P = 0.0186), and in the rate at which embryos cleaved. Thus, while the median number of blastomeres per embryo was no different in the two groups at 46-49 h post insemination (three in embryos cultured in 'in-house' medium, compared with four in those cultured in commercially prepared medium; P > 0.1), the number of embryos per cycle that had cleaved to the 4-cell stage by 46-49 h post insemination was significantly greater in the Medi-Cult than in the EBSS medium (P < 0.001).

The effect of temperature fluctuations on the cytoskeletal organisation and chromosomal constitution of the human oocyte.

The effect of temperature fluctuation on spindle integrity and chromosomal organisation in the human oocyte, and the consequences of such effects on the chromosomal constitution of resulting parthenotes, were investigated. A total of 340 oocytes were stained immunocytochemically with an antibody to alpha-tubulin, and 502 were activated parthenogenetically. Exposure of oocytes to room temperature for 2, 10 or 30 min caused disruption of the spindle in 77% (n = 26), 72% (n = 18) and 89% (n = 19) of cases respectively, with evidence of chromosomal dispersal in 50%, 56% and 52.6% respectively. These effects were reversed when oocytes were returned to 37 degrees C after exposure to room temperature for 2 min, but not after 10 min or 30 min. Temperature reduction affected rates of parthenogenetic activation of oocytes (2 min: 67%, n = 27; 10 min: 68%, n = 28; 30 min: 54%, n = 35) and cleavage of resulting parthenotes, but only if oocytes were exposed to room temperature for 30 min (30 min: 53%, n = 19). There is a direct association between temperature-induced spindle damage in the oocyte (70%, 50 of 63) and chromosomal abnormalities in parthenotes developed from oocytes exposed to room temperature (56%, 23 of 41; p < 0.01).

Synthetic profiles of polypeptides of human oocytes and normal and abnormal preimplantation embryos.

There is considerable variation in the rate of development in vitro of individual preimplantation human embryos. The relationship between the rate of development and patterns of polypeptide synthesis in individual embryos was examined using SDS-PAGE and autoradiography. After incubation in [35S]methionine, 19 polypeptide bands were identified that change between fertilization and the morula stage. Although changes in two of the bands occurred in embryos that were developing normally and in ageing oocytes, and are thus independent of fertilization, the changes identified in the remaining 17 bands occurred only after fertilization. In embryos that were developing abnormally, as assessed by delayed cleavage, cleavage arrest or extensive fragmentation, the alteration in polypeptide synthetic profiles increased with increasing abnormality.

Distribution of alpha3, alpha5 and alpha(v) integrin subunits in mature and immature human oocytes.

The distribution of three integrin subunits, alpha3, alpha5 and alpha(v), in immature and mature human oocytes has been examined using immunofluorescence and confocal microscopy. The results demonstrate that both alpha5 and alpha(v) are present at the germinal vesicle stage, while alpha3 was only detected in oocytes after germinal vesicle breakdown, in metaphase I and II stage oocytes. The cortical concentration of integrin subunits alpha3 and alpha5 is consistent with their localization in the oolemma. In contrast, the homogeneous distribution of alpha(v) throughout the oocyte suggests the existence of cytoplasmic reservoirs of this protein in the oocyte.