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

Abstract

The process of micro-insemination by single or multiple sperm transfer into the perivitelline space (PVS) or by direct sperm injection into oocytes was examined by transmission electron microscopy. Spermatozoa from normal and oligozoospermic men were injected into oocytes, obtained from consenting IVF patients, mostly by zona-puncture using micromanipulators. Spermatozoa were washed by the Percoll or Ficoll methods and capacitated using Whittingham's T6 or modified Tyrode's medium or incubated in strontium medium before injection. The women were stimulated by three IVF methods and oocytes were recovered by laparoscopy or ultrasonography. Sixty-one oocytes were cultured in T6 or Ham's F-10 media (3-24 h) and were subjected to micromanipulation. Four oocytes were also studied after zona-drilling. Normal 2-pronuclear ova were developed after single-sperm transfer satisfying all morphological criteria of fertilization. Both monospermic and polyspermic fertilization resulted after multiple sperm transfer, indicating that a vitelline block to polyspermy may exist in humans. The majority of oocytes examined were unfertilized. Spermatozoa with intact or reacted acrosomes and those undergoing the acrosome reaction were found in the PVS and in the ooplasm. Abnormal spermatozoa were also seen in these locations. Quantitation of acrosomal status in 16 oocytes after multiple-sperm transfer, revealed that 24% of spermatozoa were acrosome-reacted or reacting in the PVS following Ficoll entrapment, while 76% of spermatozoa were intact (33% of these abnormal). Sperm transfer seemed to be the least invasive, while direct sperm injection was comparatively destructive to oocytes. Drilling with acid made larger breaches in the zona when compared with mechanical perforation and spermatozoa occasionally escaped through breaches. Three 2-pronuclear ova obtained after multiple sperm transfer have resulted in two pregnancies, in cases of severe oligozoospermia, during the course of this study.