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In Vitro Fertilization of Hamster Ova as an Aid in Assessing the Fertilizing Capacity of Human Spermatozoa
Thesis Abstract:
Studies have been recently undertaken in many research laboratories to examine the fertilizing capacity of spermatozoa from human males with histories of unexplained infertility to investigate their ability to penetrate ova. Since human ova were not readily available, hamsters were used as a substitute for these studies and there is ongoing research to test the validity of this assay as a reliable diagnostic tool for examining sperm-ovum interaction in man and animals.
A study was undertaken to develop such an assay system for evaluating the causes of "unexplained infertility" in males in the National Population and Family Development Board in Kuala Lumpur.
The semen characteristics of 175 ejaculates from 35 donoes (one ejaculate per donor per week) showed 10 fertile patients to possess normal semen parameters and 25 infertile patients to have azoospermia (4%), oligoospermia (44%), poor semen counts (32%), normozoospermia (12%), and polyzoospermia (8%). Ten of the infertile patients with semen parameters within normal limits were put to the sperm penetration assay.
Thirty mature cycling female golden hamsters (Mesocricetus auratus) were superovulated using 30 IU pregnant mare's serum gonadotropin on the day following estrus which was followed 48 hours later by 30 IU human chorionic gonadotropin (HDG), with both drugs being administred intraperitoneally.
Uterine and oviductal flushings taken 15-17 hours after the injection of HCG gave 30±1.03 ova (20-43) for each hamster.
Ham's F10 medium was found to be suitable for maintainance of ova and in vitro fertilization. To obtain zona-free ova, the cumulus cells and zona pellucida were removed with 0.1 percent hyaluronidase for 5-6 mins at 37ºC and 0.1 percent trypsin for 2-3 mins at 37ºC, respectively. Eighty percent of zona-free ova were fertilized with sperm (1.5 x 106 sperm/ml) from fertile donors, when the semen was washed with Ham's F10 medium and pre-incubated at 37ºC for six hours (P<0.05).
Penetration rates were significantly lower (P<0.01) for zona-intact and cumulus-intact ova at sperm pre-incubation times of 3-9 hours. When sperm pre-incubation was fixed at six hours, maximum penetration rates of 80.0±2.9 percent were observed when zona-free ova were allowed to interact with sperm for six hours (P<0.05). Penetration rates in zona-intact and cumulus-intact ova were significantly lower (P<0.01) than zona-free ova when sperm-ovum interaction was six hours. Penetration of zona-free ova was not observedd in 60 percent of "infertile" patients with normal semen characteristics, even when ejaculates per week per patient were used over a five-week period.
Chromosome analysis was possible in 95 percent of penetrated ova and the presence of discrete haploid sets of human sperm chromosomes in the hamsster ooplasm was used to confirm penetration.
The results of this study demonstrated that the zona pellucida of the hamster ovum acts as a barrier to human sperm and zona-free hamster ova may be a suseful substitute for human ova to test the fertilizating capabilities of spermatozoa taken from males with history of unexplained infertility.