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.

Experience with zona drilling and zona cutting to improve fertilization rates of human oocytes in vitro

“…Polyspermy rates of 12 and 18% have been reported following partial zona dissection (PZD) compared with polyspermy rates in control oocytes of 20 and 29%, respectively (Maker and Cohen, 1989;Cohen et al, 1989). Sathananthan et al (1989) and Fishel et al (1990) obtained monospermic fertilization after multiple spermatozoa were transferred into the perivitelline space of human oocytes. We have observed motile spermatozoa swimming in the perivitelline space of normally fertilized ZC oocytes-a phenomenon similar to that seen in fertilized rabbit oocytes where there exists a very strong vitelline block to polyspermy (Yanagimachi, 1988) We have also observed normozoospermic fertilization in all six ZC and six intact oocytes from one patient, indicating that sperm function was normal and a vitelline block to polyspermy was operating.…”

Centrioles in the beginning of human development.

“…The proximal centriole consists of nine triplets of MTs showing the typical 9 + 0 organization and is associated with osmiophilic centrosomal material. After gamete fusion, the sperm midpiece and tail are invariably incorporated into the ooplasm, and the centriolar region often remains attached to the decondensing sperm nucleus and persists after male pronuclear formation (16)(17)(18). This study demonstrates the presence of centrioles associated with centrosomes in the first mitotic spindle of the human fertilized oocyte.…”

“…The association of these centrioles with the spindle does not seem to be accidental as there was always a close relationship between the sperm flagellar neck or midpiece with the male pronucleus during its formation (16,17) and later during its association with the female pronucleus (18,21). It is possible, although unlikely, that the paternal centriole may associate secondarily with spindle poles at syngamy after pronuclear disorganization.…”

“…(i) The proximal sperm centrioles ( Fig. 1) are closely associated with the male pronucleus during its entire maturation (16)(17)(18). (ii) The structure of the centrioles observed at spindle poles in normal monospermic embryos (Fig.…”

Induction of Acrosome Reaction in Human Sperm by Exposing to an Electrical Field

“…The decrease in motility of sperm at the higher voltages implies that, apart from inducing acrosome reaction in those sperm that are susceptible, damage to the motility apparatus and possibly the hsiogenic segment has occurred. Keeping this in mind and also the finding of Sathananthan et al [21] that sperm midpiece contributes a centriole role to the first mitotic spindle, further investigation, such as intracytoplasmic injection of these sperm into the hamster oocytes and karyotyping of decondensed sperm head, needs to be carried out.…”