Michael Femi Obasaju

Sperm quality may adversely affect the chromosome constitution of embryos that result from intracytoplasmic sperm injection.

OBJECTIVE To determine whether the high rate of chromosomal abnormalities in the embryos of an infertile couple were caused by a paternal factor that may have involved the sperm centriole. DESIGN Case report. SETTING Private IVF program. PATIENT(S) An infertile couple who underwent IVF-ET because of severe male factor infertility and endometriosis. INTERVENTION(S) Preimplantation genetic diagnosis of chromosomal abnormalities in embryos derived from two cycles of ICSI in which the husbands sperm was used and one in which donor sperm was used. MAIN OUTCOME MEASURE(S) Preimplantation genetic diagnosis with fluorescence in situ hybridization using probes for chromosomes X, Y, 13, 16, 18, and 21, and determination of hCG levels. RESULT(S) Most of the embryos derived from the cycles in which the husbands sperm was used were chromosomally abnormal (82%), whereas all the embryos derived from the cycle in which donor sperm was used were chromosomally normal. The cycle in which donor sperm was used resulted in an ongoing pregnancy. CONCLUSION(S) Paternal factors, which most likely derive from the centrosome, can contribute to numerical chromosomal abnormalities, which in turn may predispose to implantation failure.

Pregnancies from single normal embryo transfer in women older than 40 years

The aim of this retrospective study was to determine the pregnancy rate from the transfer of single genetically normal embryos in patients of advanced reproductive age. The study group included 23 patients (mean age 42.2 +/- 1.3 years) who underwent 27 in-vitro fertilization (IVF) cycles in which preimplantation genetic diagnosis (PGD) was carried out on single blastomeres from day 3 embryos. The control group included 54 patients (mean age 43.3 +/-1.9 years) who underwent 69 cycles of IVF without PGD. Ovarian stimulation in all patients consisted of follicular phase leuprolide acetate administration, followed by ovulation induction with gonadotrophins. The mean number of biopsied embryos was 5.6 +/- 0.5. No embryo transfer occurred in six patients (10 cycles) because all embryos biopsied were abnormal. Seventeen patients (17 cycles) each had one genetically normal embryo transferred resulting in six on-going clinical pregnancies (35% per embryo transfer cycle). The mean number of embryos transferred in the control group was 4.0 +/- 0.8. Nineteen clinical pregnancies were obtained in 69 transfer cycles in the control group (28% per embryo transfer cycle). The transfer of a single normal embryo in patients of advanced reproductive age can lead to acceptable pregnancy rates. Aneuploidy appears to be a major cause of reproductive failure in this group of patients.

An Assay Using Embryo Aggregation Chimeras for the Detection of Nonlethal Changes in X-Irradiated Mouse Preimplantation Embryos

We have developed a short-term in vitro assay for the detection of sublethal effects produced by very low levels of ionizing radiation. The assay utilizes mouse embryo aggregation chimeras consisting of one irradiated embryo paired with an unirradiated embryo whose blastomeres have been labeled with fluorescein isothiocyanate (FITC). X irradiation (from 0.05 to 2 Gy) and chimera construction were performed with four-cell stage embryos, and the chimeras were cultured for 40 h to the morula stage. The morulae were partially dissociated with calcium-free culture medium and viewed under phase contrast and epifluorescence microscopy to obtain total embryo cell number and the cellular contribution of irradiated (unlabeled) and control (FITC labeled) embryos per chimera. In chimeras where neither embryo was irradiated, the ratio of the unlabeled blastomeres to the total number of blastomeres per chimera embryo was 0.50 (17.8 +/- 5.6 cells per unlabeled embryo and 17.4 +/- 5.5 cells per FITC-labeled partner embryo). However, in chimeras formed after the unlabeled embryos were irradiated with as little as 0.05 Gy, the ratio of unlabeled blastomeres to the total number of blastomeres per chimera embryo was 0.43 (P less than 0.01). The apparent decreases in cell proliferation were not observed in irradiated embryos that were merely cocultured with control embryos, regardless of whether the embryos were zona enclosed or zona free. We conclude that very low levels of radiation induce sublethal changes in cleaving embryos that are expressed as a proliferative disadvantage within two cell cycles when irradiated embryos are in direct cell-to-cell contact with unirradiated embryos.

Effects of phlorizin and ouabain on the polarity of mouse 4-cell/16-cell stage blastomere heterokaryons

Cell polarity is thought to be required for the efficient production of nascent blastocoele fluid, which begins at the 16-cell stage of mouse preimplantation development. In this study the 4-cell/16-cell blastomere heterokaryon was used to test the hypothesis that solute transport across the apical membrane domain induces the apical-basal axis of organelle distribution across polar 16-cell-stage blastomeres. Fusion of 4-cell/16-cell blastomere pairs resulted in a population of heterokaryons of which 65% were polar (contain an apical plasma membrane domain from a polar 16-cell-stage plasma membrane insert) and 30% were apolar (contain an apolar 16-cell-stage plasma membrane insert). Polar heterokaryons were distinguished from apolar ones by labeling their apical domains with fluorescent succinylated concanavalin A. In polar heterokaryons, both nuclei (labeled with Hoeschst 33242) were immediately subjacent to the apical plasma membrane domain, while in apolar heterokaryons both nuclei were located centrally. Two inhibitors of apical transmembrane solute transport--phlorizin, which inhibits brush border (apical) Na+/glucose symporters, and ouabain, which inhibits Na+/K+-ATPase, thereby modifying the transmembrane Na+ gradient--were examined for their effect on nuclear position in polar and apolar heterokaryons after a 4-hr incubation in either inhibitor. Both ouabain (L.M. Wiley, 1984, Dev. Biol. 105, 330-342) and phlorizin (this study) had a biphasic effect on the rate of nascent blastocoele fluid accumulation such that at lower concentrations (ouabain, 10(-5) M; phlorizin, 10(-6) M) fluid accumulation was accelerated and at higher concentrations (both inhibitors, 10(-4) M) fluid accumulation was delayed. In polar heterokaryons, both concentrations of each inhibitor caused the nuclei to become displaced basally from their normal location against the apical plasma membrane domain. Both nuclei, however, remained on the axis of polarity passing through the apical domain. The magnitude of displacement was greater at higher concentrations of either inhibitor. Neither inhibitor affected nuclear position in apolar heterokaryons. These observations agree with the hypothesis that apical plasma membrane solute transport maintains the asymmetric organelle distribution across the apical-basal axis of polar 16-cell-stage blastomeres.

Immunofluorescent localization of immunoglobulins on the cell surface of mouse oocytes and preimplantation embryos

Living mouse oocytes and preimplantation embryos were assayed by indirect immunofluorescence for their ability to adsorb heterologous serum proteins from culture media to their cell surfaces. Bovine and human immunoglobulins of the IgG class were adsorbed by the oocytes and all stages of preimplantation embryos, while IgG of mouse or goat origin was not. In contrast none of the serum albumins was adsorbed to the cell surfaces of mouse oocytes and preimplantation embryos. From the differential binding of IgG of some, but not all, of the species that were tested, we concluded that these cell surface IgG “receptors” on mouse oocytes and preimplantation embryos are likely to be heterophilic in nature. Similar observations were made irrespective of the strain of mice used to provide the oocytes and embryos. These observations raise the question of whether human oocytes and preimplantation embryos should also be assayed for their ability to adsorb animal serum proteins that are possible candidates as a substitute protein supplement for human serum in culture medium used in human in vitro fertilization/embryo transfer programs.

Reproductive effects of chlorpromazine exposure to female mice: Cell proliferation disadvantage revealed by the chimera embryo assay

Administration of chlorpromazine-HCl at 5 to 15 mg/kg bodyweight to pregnant CD-1 mice at 24 h after human chorionic gonadotropin (hCG) (20-23 h after mating) inhibited blastocyst formation and reduced the cell number of embryos recovered at 95 h after hCG. When embryos are recovered at the two- to four-cell stage (48-50 h after hCG) and cultured for an additional 47 h (to 95 h after hCG) or 72 h (to 120 h after hCG), blastocyst formation and embryo cell number were similarly reduced. When the dose range was reduced to 0.5 to 2 mg/kg bodyweight, no significant effect of the drug was observed on blastocyst formation or on embryo cell number. However, when aggregation chimeras were formed between embryos recovered from drug-exposed females and from untreated females, a decrease in cell proliferation rate of the embryo from the drug-exposed female was observed at a dose of 2 mg/kg bodyweight. This result indicates that exposing pregnant mice to chlorpromazine-HCl at doses as low as 2 mg/kg bodyweight can induce a potential for decreased cleavage rate in their pre-implantation embryos that can be revealed by challenging those embryos by direct contact with embryos from nonexposed females. Finally, when four-cell stage embryos recovered from untreated females cultured in the presence of chlorpromazine (0.1-25 mM), blastocyst formation and embryo cell number were significantly reduced in a dose-dependent manner. This last result suggests that in vivo the drug may act directly on the embryo from the pronuclear stage to the early morula stage of development.

Pregnancy Rate from the Transfer of a Single Normal Embryo in Women of Advanced Reproductive Age Undergoing PGD for Determination of Aneuploidy

MII n 5 203 46 (22.7%) 41 (20.2%) 40 (19.7%) 33 (16.3%) 91 (44.9%) GV n 5 93 6 (6.5%)* 3 (3.2%)* 8 (8.6%)† 4 (4.3%)* 18 (19.3%)* *P ,0.05 compared to MII oocytes. †P 50.06 compared to MII oocytes. The data shows that there was a significant difference (P,0.05, Chi Square Test) between the frequency of aneuploidy in MII and GV oocytes for chromosomes 11, 17 and 22 and was bordering on significance for chromosome 16. There was a significant difference (P,0.05, Chi Square Test) in the number of GV oocytes and MII oocytes that were aneuploid for at least one of the four chromosomes. Conclusions: We have shown that aneuploidy occurs in GV oocytes at a frequency of approximately 6% for each of the chromosomes tested, and that almost 20% of GV oocytes have an aneuploidy. The frequency of aneuploidy was significantly greater in MII oocytes confirming that many aneuploidies also occur during the first meiotic division. Importantly, it has also shown that a significant number of oocytes are aneuploid even prior to metaphase I.

Detection of antisperm antibodies: their localization to human sperm antigens that are transferred to the surface of zona-free hamster oocytes during the sperm penetration assay**Supported by National Institutes of Health grants HD-15149 and HD-19587.

The authors have developed an extension of the sperm penetration assay for detecting serum immunoglobulins to sperm antigens that are transferred to the plasma membrane of a sperm-penetrated hamster oocyte. After the hamster oocytes have been scored for sperm penetration by observing for the presence of swollen sperm heads, they are incubated in serum followed by either a 20-minute treatment with rhodamine-conjugated protein A (which binds to most subclasses of IgA, IgG, and IgM) or a 2-hour incubation in guinea pig serum (complement). Positive fluorescence indicates that the serum contains antibodies to sperm antigens that were transferred to the surface of an oocyte during gamete fusion. Complement-mediated lysis indicates that the immunoglobulin that is bound can also fix complement. The advantages of this assay for detection of serum antisperm antibodies are that it is an extension of a widely used assay, is rapid and requires readily available reagents and equipment, can detect most subclasses of IgA, IgG, and IgM, detects antibodies to those sperm antigens that may be transferred to the oocyte during fertilization, and indicates whether the detected antisperm antibodies can mediate complement-dependent lysis of the fertilized oocyte.