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Research Paper On Human Fertilization

Abstract

This study compares failed fertilization oocytes from patients participating in an in-vitro fertilization (IVF) programme with failed fertilization oocytes from B6SJLF1/J mice, in order to characterize and describe the distribution of DNA in oocytes that do not undergo normal fertilization. Our goal is to evaluate the mouse IVF system as a model to gain insight into reasons for human fertilization failures. All oocytes were stained with the vital fluorescent dye, Hoechst 33342, which rapidly stains double-stranded DNA. Of the 237 human oocytes that had been scored as failed fertilization by brightfield microscopy, 61 (25.7%) showed the presence of at least one spermatozoon within the oocyte cytoplasm. In contrast, out of 69 failed fertilization mouse oocytes, only one oocyte showed the presence of a spermatozoon within its cytoplasm. Mouse failed fertilization oocytes exhibited a significantly lower internal sperm rate (P < 0.0001) than human failed fertilization oocytes. Human failed fertilization oocytes show a higher incidence of sperm penetration, but the cytoplasm fails to support pronuclear development, whereas, at least in this strain, mouse failed fertilization oocytes arise from an inability of the spermatozoa to penetrate the oocyte. This study suggests that the mouse is not a clinically relevant model for human fertilization failures.

fertilization failure, Hoechst 33342 dye, human and mouse IVF

Introduction

Normal fertilization results in the formation of a zygote from the union of an egg with a single spermatozoon. Under normal conditions, sperm decondensation and pronucleus formation occur as a continuum. Time course studies in both human and rodents show that the spermatozoon's nuclear decondensation/recondensation, meiotic progression, and formation of both the male and female pronucleus occur in a co-ordinated fashion (Perreault et al., 1987; Wright and Longo, 1988; Lassalle and Testart, 1991; Yanagimachi, 1994; Payne et al., 1997). Failure of these events to occur at any stage results in the inability of the oocyte to develop normally. In the in-vitro fertilization (IVF) clinic, the absence of two pronuclei in human eggs 16–18 h after insemination is taken as evidence that normal fertilization has not taken place. Fertilization failure of all or even just some of a patient's retrieved oocytes is a frustrating experience for both the patient and the IVF team, and could account for some sources of undiagnosed human infertility. The observation of only one (or no) pronucleus indicates that normal fertilization has not taken place, but gives limited information about the step at which the block to fertilization occurred. The Hoechst dye 33342 was used throughout this study to detect sperm nuclei that had penetrated the oocyte but failed to form a pronucleus. Hoechst dyes are non-toxic, supra-vital fluorescent dyes which specifically bind the adenine and thymidine bases of double-stranded DNA (Latt and Stetten, 1976; Ardent-Jovin and Jovin, 1977; Luttmer and Longo, 1986). Staining failed fertilization oocytes with Hoechst 33342 can provide valuable clinical information as to the cause of failed fertilization (Urner et al., 1993; Van Blerkom et al., 1994; Asch et al., 1995; Flaherty et al., 1995; Dubey et al., 1997).

In light of the moral, ethical, and legal issues involved in working with human gametes, as well as their limited availability, it would be very useful to have an appropriate animal model in which to study the reasons for human fertilization failures. The murine IVF system and embryo culture is widely used as a model for human IVF (Martin-DeLeon, 1989; Alsalili et al., 1997; Janssenswillen et al., 1997; Matson et al., 1997). However, differences may exist between the two species that would make the mouse system a poor model, specifically with regard to methods of centrosome inheritance. During human fertilization the spermatozoon restores the zygotic centrosome (Simerly et al., 1995), whereas the mouse follows a maternal method of centrosome inheritance (Schatten et al., 1985, 1986). This study was undertaken to characterize and describe the distribution of DNA in human oocytes that have failed to undergo normal fertilization and to attempt to evaluate the mouse IVF system as a model to gain insight into reasons for human fertilization failures.

Materials and methods

The method of ovulation augmentation used for all patients in this study was a flare-up protocol using leuprolide acetate as previously described (Schalkoff et al., 1993). Oocytes from IVF patients were retrieved with vaginal ultrasound-guided aspiration. The oocytes were cultured in micro-drops under mineral oil containing human tubal fluid (HTF; Irvine Scientific, Santa Ana, CA, USA) and 10% human serum albumin (plasmanate; Bayer Pharmaceutical, Elkheart, IN, USA) for 4–6 h prior to insemination. Semen samples were all prepared using a discontinuous Percoll preparation with gradient and centrifugation parameters calculated individually for each sample. Eggs were inseminated with 0.5 or 1.50×105 motile spermatozoa per micro-drop, and assessed 16–18 h later for evidence of fertilization. Zygotes containing two pronuclei were moved to fresh medium and cultured for an additional 24 h prior to the embryo transfer. Only those oocytes that had not undergone normal fertilization were used in this study.

A total of 63 patients, all having signed informed consent, with 237 failed fertilization oocytes were included in this study. All IVF procedures with oocytes that had failed to undergo normal fertilization were included in this study, and no selection of oocytes based on type of infertility was made. All forms of infertility treated at Boston IVF were part of this study, including male factor, tubal factor, secondary infertility and some unexplained infertility. No oocytes from intracytoplasmic sperm injection (ICSI) procedures were included in this study.

B6SJLF1/J mice, purchased from Jackson Laboratory, Bar Harbor, ME, USA, were used for all experiments. Ovulated eggs were collected from 6–12 week old females. Ovulation was induced by i.p. injection of 7.5 IU of pregnant mare's serum (PMS, Sigma, St Louis, MO, USA) and 7.5 IU of human chorionic gonadotrophin (HCG, Sigma), 48 h apart. Cumulus-cell complexes were collected from the ampullar oviduct, 15–17 h post-HCG into an organ culture dish (Falcon; Allegiance, McGraw Park, IL, USA) containing non-bicarbonated HTF supplemented with 10% plasmanate.

Males were caged individually for at least 3 weeks prior to removing intact caudal epididymides. Spermatozoa were manually squeezed from the epididymis using a 30G needle and tweezers. The spermatozoa were kept in an organ culture dish containing 1 ml of HTF medium at 37°C in an atmosphere of 5% CO2 for 1½ h, to allow capacitation to take place. Cumulus enclosed oocytes were inseminated with 1×106 spermatozoa/ml, in 100 μl drops under mineral oil. Insemination took place 1–1½ h post-removal of the oocytes from the oviduct. The gametes were incubated at 37°C and 5% CO2.

Oocytes were incubated for 10 min at 37°C, in human tubal fluid medium supplemented with 0.5% bovine serum albumin and 10 μg Hoechst-33342 dye (bisbenzimide trihydrochloride, Sigma). Oocytes were then placed on a coverslip for viewing. Oocytes were examined using a Zeiss ICM 405 fluorescent microscope with a 100 W arc bulb for epifluorescence. The DNA + H-33342 complex was excited with a 355 nm UV light and epifluorescence emission of 465 nm was viewed and photographed. A G 365 excitation filter, an FT 395 dichromatic beam splitter and an LP 420 barrier filter were used. Both epifluorescent and brightfield photographs were taken using a Sony video graphic printer (UP-870MD), or the video frame grabber function of the IPLab Spectrum Software (Scanalytics Inc., Fairfax, VA, USA). Analysis of chromatin was performed either manually on the Sony photographs or using NIH Imaging software (Research Service Branch of the National Institute of Mental Health, Bethesda, MD, USA) on the video frame grabbed computer images. The chromatin organization of the maternal and paternal genomes were determined from analysis of the fluorescent images.

Results

Maturation status of failed fertilization oocytes

A total of 237 human eggs (mean 8.3 per patient) that had been scored as failed fertilization via brightfield microscopy were examined via fluorescent microscopy after Hoechst 33342 labelling in vitro. None of these oocytes had undergone normal fertilization and none was suitable for embryo transfer. The maternal genome was assessed, and the majority (191/237, 80.6%) of the human failed fertilization oocytes were in the metaphase II (MII) PB stage of development. These oocytes contained maternal DNA and a polar body, which under fluorescence showed the presence of double-stranded DNA. The remaining oocytes came under the following categories: germinal vesicle (1/237, 0.4%), germinal vesicle breakdown/ metaphase I (MI) (12/237, 5.1%), one pronucleus with no visible pronuclear membrane under brightfield microscopy (24/237, 10.1%) and fragmented (9/237, 3.8%) (Table I).

The majority of mouse failed fertilization oocytes were also observed to be mature (59/69, 85.5%). Mouse failed fertilization oocytes were observed in the following categories: germinal vesicle (0/69, 0%), MII PB (32/69, 46.4%), MII no PB (27/69, 39.1%), one pronucleus with no visible pronuclear membrane under brightfield microscopy (5/69, 7.2%) and fragmented (5/69, 7.2%) (Table I). It has been noted that typically the first polar body degenerates during the first cell cycle in embryos derived from F1 mouse eggs (Howlett and Bolton, 1985). Donahue (1972) also observed that most first polar bodies are degenerated by the time of sperm penetration in CF1 mice. To confirm that MII oocytes lacking a PB were in fact mature in our system, a mouse IVF experiment was performed. Oocytes were collected from stimulated B6SJLF1/J mice. The cumulus cells were removed with hyaluronidase and the oocytes were separated into two groups, based on the presence or absence of a visible PB using the brightfield microscope. Only 27 out of 72 oocytes contained a visible polar body. Both groups were inseminated and scored for 2-cell stage development at 24 h post-insemination. Out of the 45 oocytes that showed no visible polar body at time of collection, 40 were scored as 2-cell embryos. Of those embryos, 35 went on to develop into blastocysts 5 days post insemination. Figure 1 demonstrates that most eggs collected from the oviduct of B6SJLF1/J mice are in fact mature (MII), with a high percentage (62.5%, 45 out of 72) of these eggs having undergone first polar body degeneration prior to sperm penetration. These results show that mouse oocytes with no visible polar body can undergo normal 2-cell and subsequent blastocyst development.

Rate of sperm penetration

Of the 237 human oocytes that were scored as failed fertilization by brightfield microscopy, 61 (25.7%) showed the presence of at least one spermatozoon within the oocyte cytoplasm using the Hoechst dye. Specifically, the failed fertilization oocytes from patients who had at least a 50% normal fertilization rate were used in this study. In this sub-group of 42 oocytes, 15 contained an internal spermatozoon (35.7%). The spermatozoa in these oocytes penetrated the zona pellucida and entered the oocyte's cytoplasm but arrested prior to reaching the two pronuclear stage of development. In contrast, out of 69 mouse oocytes scored as failed fertilization by brightfield microscopy, only one oocyte showed the presence of a spermatozoon within its cytoplasm using the Hoechst dye (1.4%) (Table II). Mouse failed fertilization oocytes exhibited a significantly lower internal sperm penetration rate (P < 0.0001).

Discussion

Time-course studies in both human and rodents show that sperm nuclear decondensation/recondensation, meiotic progression, and formation of both the male and female pronucleus occurs in a co-ordinated fashion. It would be expected that failed fertilization oocytes arising from IVF from both mouse and humans might arrest at similar stages of development. However, the results presented here highlight observable differences between failed fertilization oocytes from human and mouse IVF systems, and suggest caution in using the mouse failed fertilization oocytes as a model in which to study human fertilization failures.

In both populations, the majority of failed fertilization oocytes were mature, 80.6 and 85.7% for human and mouse, respectively. At least in this strain of mice (B6SJLF1/J), first polar body degeneration appears to be more prevalent than in human oocytes.

In comparison with mouse, human failed fertilization oocytes show a higher rate of sperm penetration of the plasma membrane, followed by developmental arrest prior to pronuclear development. Human failed fertilization oocytes from patients with greater than 50% normal fertilization rates have a 35.7% sperm penetration rate with subsequent developmental arrest prior to pronuclear formation. Human failed fertilization oocytes have been assessed in several cytogenetic studies, and it was shown that 8.2% (Wall et al., 1996) and 32.3% (Edirisinghe et al., 1997) of human failed fertilization oocytes contained sperm chromosomes. In contrast, if penetration occurs in failed fertilization mouse oocytes there is only a 1.4% rate of the spermatozoa arresting in development prior to 2-cell formation. These observations suggest that mouse failed fertilization oocytes arise from an inability of the spermatozoa to penetrate the oocyte, whereas human failed fertilization oocytes arise from an inability of the cytoplasm to support pronuclear development despite a higher rate of sperm penetration. This could suggest that human spermatozoa more easily penetrate the zona pellucida, but that there is an additional block present in human oocyte cytoplasm that prevents some spermatozoa from developing into functional pronuclei.

Dozortsev et al. (1997) suggested that in some cases, failure of oocyte activation after human ICSI could be due to the relative deficiency of sperm-associated oocyte activating factor (SAOAF) in the selected spermatozoa. It is possible that some human failed fertilization oocytes are the result of a deficiency of activating factors in the spermatozoa; however, this was not specifically investigated in our study. It has also been postulated that failure of fertilization after human ICSI may be due, in part, to poor chromatin packaging and/or damaged DNA of the penetrated spermatozoa (Sakkas et al., 1996).

It is unlikely that the block to pronuclear development in our system is due to cytoplasmic immaturity, since this appears to be associated with an increase in premature chromosome condensation (PCC) after IVF (Calafell et al., 1991). PCC was not observed in the human failed fertilization oocytes. Furthermore, evidence has been provided that the gonadotrophin-releasing hormone analogue/human menopausal gonadotrophin (GnRHa/HMG) stimulation results in an increase in the number of mature failed fertilization oocytes observed over the use of other stimulation protocols (Pieters et al., 1991).

A potential bias in the results of this study could be the use of epididymal spermatozoa in the mouse IVF system and the use of ejaculated spermatozoa in the human system. Normal fertile mouse offspring have been obtained after ICSI of not only mature (epididymal) and immature (testicular) but also round spermatids, suggesting that genomic imprinting of the male germ cell is complete before spermiogenesis in the mouse (Yanagimachi, 1998). Furthermore, the high fertilization rates using mouse epididymal spermatozoa in IVF (control 2-cell rate of 73.6%) would also suggest that these spermatozoa are mature and can be compared to IVF results using ejaculated spermatozoa.

The observations presented here suggest that in mice, the failed fertilization oocytes arise from an inability of the spermatozoa to penetrate the oocyte, whereas in humans, the failed fertilization oocytes have a higher incidence of sperm penetration but the cytoplasm fails to support pronuclear development. This study highlights observable differences between failed fertilization eggs from human and mouse IVF systems and suggests that the mouse is not a clinically relevant model for human fertilization failures.

Table I.

Nuclear status of maternal chromatin from both human and mouse failed fertilization oocytes

Stage No. of failed fertilization oocytes observed (%) 
Human Mouse 
Germinal vesicle  1 (0.4)  0 (0) 
GVBD/MI/MII no PB  12 (5.1) 27 (39.1) 
MII PB 191 (80.6) 32 (46.4) 
PN  24 (10.1)  5 (7.2) 
Fragmented  9 (3.8)  5 (7.2) 
Stage No. of failed fertilization oocytes observed (%) 
Human Mouse 
Germinal vesicle  1 (0.4)  0 (0) 
GVBD/MI/MII no PB  12 (5.1) 27 (39.1) 
MII PB 191 (80.6) 32 (46.4) 
PN  24 (10.1)  5 (7.2) 
Fragmented  9 (3.8)  5 (7.2) 

View Large

Table II.

Sperm penetration in mouse compared with human oocytes after failed fertilization

NF oocytes with internal spermatozoa (%) Overall sperm penetration rates (%) 
Mouse  1/69 (1.4) 191/265 (72) 
Human 15/42 (35.7) 132/159 (83) 
NF oocytes with internal spermatozoa (%) Overall sperm penetration rates (%) 
Mouse  1/69 (1.4) 191/265 (72) 
Human 15/42 (35.7) 132/159 (83) 

View Large

Figure 1.

Distribution of mouse oocytes collected from stimulated B6SJLF1/J mice and their subsequent development to the 2-cell and blastocyst stage. Prior to insemination, oocytes were separated into two groups based on the presence (MII PB) or absence (MII no PB) of a visible polar body using brightfield microscopy. Blastocyst values are expressed as a percentage of 2-cell stage embryo development. Control values were derived from six separate experiments using mouse epididymal spermatozoa.

Figure 1.

Distribution of mouse oocytes collected from stimulated B6SJLF1/J mice and their subsequent development to the 2-cell and blastocyst stage. Prior to insemination, oocytes were separated into two groups based on the presence (MII PB) or absence (MII no PB) of a visible polar body using brightfield microscopy. Blastocyst values are expressed as a percentage of 2-cell stage embryo development. Control values were derived from six separate experiments using mouse epididymal spermatozoa.

This work was supported in part by a grant from Boston Fertility Laboratories to R.D.Powers. The authors gratefully acknowledge Drs G.Schatten and C.Simerly for their helpful discussions.

References

Alsalili, M., Thornton, and Fleming, (

1997

) The effect of the anaesthetic, propofol, on in vitro oocyte maturation, fertilization and cleavage in mice.

Hum. Reprod.

 ,

12

,

1271

–1274.

Google Scholar

Ardendt-Jovin, D. and Jovin, T. (

1977

) Analysis and sorting of living cells according to deoxyribonucleic acid content.

J. Histochem.

 ,

25

,

585

–589.

Google Scholar

Asch, R., Simerly, C., Ord, T. et al. (

1995

) The stages at which human fertilization arrests: microtubule and chromosome configurations in inseminated oocytes which failed to complete fertilization and development in humans.

Hum. Reprod.

 ,

10

,

1897

–1906.

Google Scholar

Calafell, J.M., Badenas, J., Egozcue, J. and Santalo, J. (

1991

) Premature chromosome condensation as a sign of oocyte immaturity.

Hum. Reprod.

 ,

6

,

1017

–1021.

Google Scholar

Donahue, R. (

1972

) Fertilization of the mouse oocyte: sequence and timing of nuclear progression to the two-cell stage.

J. Exp. Zool.

 ,

180

,

305

–318.

Google Scholar

Dozortsev, D., Qian, C., Ermilov, A. et al. (

1997

) Sperm-associated oocyte-activating factor is released from the spermatozoon within 30 min after injection as a result of the sperm–oocyte interaction.

Hum. Reprod.

 ,

12

,

2792

–2796.

Google Scholar

Dubey, A., Pennies, A., Emmi, A. et al. (

1997

) Failed fertilization after intracytoplasmic sperm injection: the extent of paternal and maternal chromatin decondensation.

Fertil. Steril.

 ,

68

,

714

–717.

Google Scholar

Edirisinghe, W.R., Murch, A., Junk, S. and Yovich, J.L. (

1997

) Cytogenetic abnormalities of unfertilized oocytes generated from in-vitro fertilization and intracytoplasmic sperm injection: a double blind study.

Hum. Reprod.

 ,

12

,

2784

–2791.

Google Scholar

Flaherty S.P., Payne, D., Swann, N.J. and Matthews, C.D. (

1995

) Aetiology of failed and abnormal fertilization after intracytoplasmic sperm injection.

Hum. Reprod.

 ,

10

,

2623

–2629.

Google Scholar

Howlett, S.K. and Bolton, V. (

1985

) Sequence and regulation of morphological and molecular events during the first cell cycle of mouse embryogenesis.

J. Embryol. Exp. Morphol.

 ,

87

,

175

–206.

Google Scholar

Janssenswillen, C., Christiaens, F., Camu, F. and Van Steirteghem, A. (

1997

) The effect of propofol on parthenogenetic activation, in vitro fertilization and early development of mouse oocytes.

Fertil. Steril.

 ,

67

,

769

–774.

Google Scholar

Lassalle, B. and Testart, J. (

1991

) Sequential transformations of human sperm nucleus in human eggs.

J. Reprod. Fertil.

 ,

91

,

393

–402.

Google Scholar

Latt, S.A. and Stetten, G. (

1976

) Spectral studies on 333258 Hoechst and related bisbenzimidazole dyes useful for fluorescent detection of deoxyribonucleic acid synthesis.

J. Histochem.

 ,

24

,

24

–33.

Google Scholar

Luttmer, S. and Longo, F. (

1986

) Examination of living and fixed gametes and early embryos stained with supra vital fluorochromes (Hoechst 33342 and, 3,3′-dihexyloxacarbocyanine Iodine).

Gamete Res.

 ,

15

,

267

–283.

Google Scholar

Martin-DeLeon, P. (

1989

) Analysis of the chromosome complement in outbred mouse sperm fertilized in vitro.

Gamete Res.

 ,

22

,

71

–81.

Google Scholar

Matson, P.L., Graefling, J., Junk, S.M. et al. (

1997

) Cryopreservation of oocytes and embryos: use of a mouse model to investigate effects upon zona hardness and formulate treatment strategies in an in-vitro fertilization programme.

Hum. Reprod.

 ,

12

,

1550

–1553.

Google Scholar

Payne, D., Flaherty, S., Barry, M. and Matthews, C. (

1997

) Preliminary observations on polar body extrusion and pronuclear formation in human oocytes using time-lapse video cinematography.

Hum. Reprod.

 ,

12

,

532

–541.

Google Scholar

Perreault, S., Naish, S. and Zirkin, B. (

1987

) The timing of hamster sperm nuclear decondensation and male pronucleus formation is related to sperm nuclear disulfide bond content.

Biol. Reprod.

 ,

36

,

239

–244.

Google Scholar

Pieters, M., Dumoulin, J., Englehart, C. et al. (

1991

) Immaturity and aneuploidy in human oocytes after different stimulation protocols.

Fertil. Steril.

 ,

56

,

306

–310.

Google Scholar

Sakkas, D., Urner, F., Bianchi, P.G. et al. (

1996

) Sperm chromatin anomalies can influence decondensation after intracytoplasmic sperm injection.

Hum. Reprod.

 ,

11

,

837

–843.

Google Scholar

Schalkoff, M.E., Oskowitz, S.P. and Powers, R.D. (

1993

) A multifactorial analysis of the pregnancy outcome in a successful embryo cryopreservation programme.

Fertil. Steril.

 ,

59

,

1070

–1074.

Google Scholar

Schatten, G., Simerly, C. and Schatten, H. (

1985

) Microtubule configuration during fertilization, mitosis and early development in the mouse and the requirements for egg microtubule-mediated motility during mammalian fertilization.

Proc. Natl Acad. Sci. USA.

 ,

82

,

4152

–4155.

Google Scholar

Schatten, H., Schatten, G., Mazia, D. et al. (

1986

) Behavior of centrosomes during fertilization and cell division in mouse oocytes and in the sea urchin eggs.

Proc. Natl Acad. Sci. USA.

 ,

83

,

105

–109.

Google Scholar

Human fertilization is a union of egg and sperm resulting in a fertilized egg, also called as zygote. Fertilization takes place inside the fallopian tube. Embryogenesis starts with fertilization of egg cell. Embryogenesis forms and develop the embryo.

Related Journals of Human Fertilization and Embryogenesis
Human Genetics and Embryology, Molecular Biology,  Journal of Medicine Microbiology and Diagnosis, Cellular and Molecular Biology, Herediatry Genetics: Current Research, Journal of embryo transfer, Journal of In Vitro Fertilization and Embryo Transfer, Iraqi Journal of Embryos and Infertility Research, Zygote, Advances in Anatomy Embryology and Cell Biology

Human Fertilization and Embryogenesis

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