Human micro-insemination by injection of single or multiple sperm: ultrastructure

The process of micro-insemination by single or muhiple spermtransfer into the perivitelline space (PVS) or by direct sperminjection into oocytes was examined by transmission electronmicroscopy. Spermatozoa from normal and oligozoospermic menwere injected into oocytes, obtained from consenting IVF patients,mostly by zona-puncture using micromanipulators. Spermatozoawere washed by the Percoll or Ficoll methods and capacitatedusing Whittingham's T₆ or modified Tyrode's medium or incubatedin strontium medium before injection. The women were stimulatedby three IVF methods and oocytes were recovered by laparoscopyor ultrasonography. Sixty-one oocytes were cultured in T₆ orHam's F-10 media (3–24 h) and were subjected to micromanipulation.Four oocytes were also studied after zona-drilling. Normal 2-pronuclearova were developed after single-sperm transfer satisfying allmorphological criteria of fertilization. Both monospermic andpolyspermic fertilization resulted after multiple sperm transfer,indicating that a vitelline block to polyspermy may exist inhumans. The majority of oocytes examined were unfertilized.Spermatozoa with intact or reacted acrosomes and those undergoingthe acrosome reaction were found in the PVS and in the ooplasm.Abnormal spermatozoa were also seen in these locations. Quantitatlonof acrosomal status in 16 oocytes after multiple-sperm transfer,revealed that 24% of spermatozoa were acrosome reacted or reactingin the PVS following Ficoll entrapment, while 76% of spermatozoawere intact (33% of these abnormal). Sperm transfer seemed tobe the least invasive, while direct sperm injection was comparativelydestructive to oocytes. Drilling with acid made larger breachesin the zona when compared with mechanical perforation and spermatozoaoccasionally escaped through breaches. Three 2-pronuclear ovaobtained after multiple sperm transfer have resulted in twopregnancies, in cases of severe oligozoo spermia, during thecourse of this study.