To assess the association of zona pellucida micromanipulation and subsequent development of monozygotic twins, cases of assisted embryo hatching (AH) and intracytoplasmic sperm injection (ICSI) were identified and related to treatment type, implantation and zygosity data. Embryology records from all patients undergoing in-vitro fertilization (IVF) at this centre from January 1995 to March 1998 were reviewed. In this study, 3546 transfer cycles were completed, with clinical pregnancy established in 1911 (54% per transfer) patients undergoing a single IVF cycle. These pregnancies occurred in 1674 (88%) IVF cycles, 120 (6%) donor oocyte cycles (DER), and 117 (6%) frozen embryo transfer (FET) cycles. During the study period, 23 cases of monozygotic (MZ) twins were identified, representing an overall frequency of 1.2%. Chorionicity was determined by transvaginal ultrasound at 7 weeks when the number of embryos transferred was less than the number of fetal heart-beats, or when >1 fetal heartbeat per gestational sac was seen. Zygosity was confirmed by placental evaluation at delivery, and corroborated the antenatal diagnosis in all cases. Among IVF study patients the frequency of MZ twinning was not statistically different between zona manipulated and zona intact subgroups. While this investigation is the largest to date describing the relationship between MZ twins and zona procedures, studies with even greater statistical power are needed to clarify it more precisely, particularly in DER and FET settings. A greater overall frequency of MZ twinning for IVF patients may be a function of the higher number of embryos transferred in IVF, rather than discrete zona manipulations.
The human zygote relies on the paternal gamete to provide the centrosome component essential for the first mitotic division. It is not known whether normal centrosome function requires an intact spermatozoon, or whether donation of an isolated paternal centrosome component can result in normal zygotes and embryos. To explore this possibility, mature human oocytes were microinjected with either intact or dissected spermatozoa. Fertilization and cleavage rates were documented; nuclear and cytoskeletal changes were observed with fluorescent immunocytochemistry; and chromosomal normality was assessed with fluorescent in-situ hybridization. A pilot study was performed to identify cytoskeletal features suggestive of centrosome function. Unfertilized oocytes and tripronucleate (3PN) zygotes from in-vitro fertilization or intracytoplasmic sperm injection were assessed to confirm the sequence of the landmarks of human fertilization. Oocytes injected with mechanically-dissected spermatozoa appear to be capable of normal pronuclear formation and embryonic cleavage, but do not undergo normal mitotic division. Although decondensed, apposed nuclei are noted in combination with diffuse cytoskeleton assembly, no spindle was detected in any zygote resulting from the injection of a dissected spermatozoon. Analysis of selected embryos resulting from dissected sperm injection revealed chromosomal mosaicism in the majority of specimens. The lack of a bipolar spindle, in combination with chromosomal mosaicism, suggests abnormalities of the mitotic apparatus when sperm integrity is impaired following dissection.
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