Role of sperm function test in failed fertilization

Fertilization in humans is the process, which culminates when the parental genomes unite

within the activated oocyte. For this to occur successfully, a series of well-synchronized

chronological events must unfold accurately. Failure of these events at any stage is lethal

for the zygote and may be the source of some undiagnosed human infertility. While sperm

behavior and the initial encounter of the gametes have been studied in humans, the motility

of the sperm and egg nuclei (male and female pronuclei) within the oocyte, which leads to

their union, is less well understood.

Today it's possible to analyze stages at which human fertilization fails with sophistication

and precision, using immunofluorescence. The role of the spermatozoon in this process can

be studied very closely and its communication with the oocyte, ones penetration occurs, can

be elucidated.

In animal species studied so far, the oocytes- activating signal uses calcium as a second

messenger. In mammalian oocytes, the source of calcium participating in these signaling

events is predominantly intracellular, although calcium influx from the extracellular space

is also involved. In human oocytes, the sperm induced- calcium oscillations can last for up

to 5-6 hrs (Sousa et al., 1996); the frequency with which individual calcium spikes appear

ranges between one spike every two minutes to one every thirty five minutes and is highly

variable from oocyte to oocyte, but relatively stable in a single oocyte during the whole

calcium oscillation period (Tesarik and Sousa, 1994; Sousa et al., 1996). In human oocytes,

the early calcium reaction to the spermatozoon is characterized by an initial group of three

to six rapidly occurring calcium spikes which is then followed by lower frequency spikes

for the rest of the calcium period (Tesarik and Sousa, 1994). Any perturbation in the correct
pattern of calcium oscillation can cause incomplete Meiotic Promoting Factor (MPF)

inactivation allowing the male nucleus to enter metaphase prematurely and generating

premature chromosome condensation (PCC).

Despite the continuous improvement of in vitro fertilization techniques, fertilization failure

is a recurrent phenomenon. This has been mainly explained in terms of chromosomal

alterations. Many years ago, several human studies that showed different types of

fertilization failures after conventional in vitro fertilization focused on oocytes analyzed by

cytogenetic techniques (Plachot and Crozet, 1992; Zenzes and Casper, 1992).

Although explanations in terms of chromosomal alterations are well-documented, the

participation of sperm decondensation, meiosis resumption, oocyte activation and pronuclei

migration has been less studied. As extensively described by Asch et al., 1995, fertilization

failure in mammals may have different causes. Firstly, the injected oocyte can fail to

initiate the biochemical processes necessary for oocyte activation (Tesarik, 1994).

Alternatively, this process may be initiated but may not occur normally, leading to an

incomplete activation. Further the spermatozoon may remain poorly accessible to oocyte

factors required for chromatin decondensation and formation of the female pronucleus

(Tesarik and Kopecny, 1989 a, b). Finally, the injection procedure may entail subtle

structural changes in oocyte organelles, not detectable by light microscopy.

In brief, in the immunoflourescence analysis zona pellucida is removed by a brief

incubation in Tyrode’s acid and the oocytes are fixed and permeabilized in a microtubule

stabilizing buffer. As a primary monoclonal antibody, anti α- acetylated tubulin is used to

identified the sperm tail, and to analyze the meiotic spindle, the material is incubated with

anti β- tubulin- Cy3 monoclonal antibodies. Chromatin is identified by counterstainig with

Hoescht 33258. Then, the processed material is mounted between a slide and a coverslips in

and examined using an epifluorescent microscope.

In order to explore the cellular and molecular aspects of fertilization failure, we studied 248

´non- fertilized´ human oocytes after in vitro fertilization (IVF) and intracytoplasmic sperm

injection (ICSI), in which no pronuclei were visualized and, in some cases, the extrusion of

the second polar body had not occurred after 20-40 hrs post insemination or injection.

In this investigation we observed that the main reason for in vitro fertilization failure after

IVF is absence of sperm penetration (54.9%, Table Ia). Microtubule and DNA analysis

revealed that 45.1% of the oocytes initiate some aspects of the fertilization process, but

arrested at specific stages. On the other hand, 13.3% of the ‘non-fertilized’ oocytes after

ICSI did not show any sperm in the ooplasm. These results are in accordance to that

previously described by Flaherty et al., 1995; Sakkas et al., 1996; Lopes et al., 1998.

The principal reason of fertilization failure after ICSI evident from this study is oocyte

activation failure (36.5%, Table Ib). Swann et al. (1990) and Dozortzev et al. (1995) have

shown that the sperm contains a temperature-sensitive cytosolic factor called oscilin, that

when injected into the oocyte is able to generate oocyte activation. The absence of this

factor or the lack of activity in ‘weak’ sperm as the one used in ICSI procedures, could

explain, at least in part, the activation failure observed after ICSI. A higher paternal DNA

fragmentation rate and an even significantly higher rate of DNA fragmentation in sperm

that failed their decondensation compared to the sperm that did not, was described by

Lopes et al. (1998). The authors suggested that a correlation exist between the rate of DNA

When sperm nuclei enter fully mature eggs they regularly transform into swelling

pronuclei, whereas those spermatozoa penetrating oocytes which were arrested at MII,

undergo conspicuous chromosome condensation. PCC was observed in 6.1% of the

oocytes studied after IVF and ICSI. This process was firstly described by Schmiady et al.

(1986), and the essential prerequisite is no activation of the oocyte and the presence of

condensing factors (e.g. MPF) in the cytoplasm preventing the sperm nuclei from

transforming into pronuclei.

Defects in pronuclear formation and/or migration has been found in a similar range after

IVF and ICSI (23.9% and 21.9%, respectively, Table Id). Schatten et al. (1994) have shown

that this situation could be possible when an inability in the assembly of the microtubules

around the paternal centrosome exists. The attraction of γ tubulins towards the centrosome

can be generated by specifics binding sites and also by energy consuming proteins like

dynein. Motility of maternal components to the paternal centrosome leads the aster

enlargement in humans, until maternal pronuclei contact occurs. During these process male

pronuclei moves towards the center of the cell to make contact to the maternal pronuclei

that migrated from it’s eccentric position near the oocyte cortex.

The overall fertilization rate gives an indication of the overall effectiveness of IVF and

ICSI in overcoming defects which prevent fertilization, but it does not provide information

on the results for individual couples or the variation in the fertilization rate in repeated

cycles. Hence, in order to provide a clear diagnosis of failed fertilization in each case, the

use of immunofluorescence is needed.

Flaherty et al., 1998, have shown that total failed fertilization is a rare event after ICSI (3%)

and is most frequent in cycles in which only one or two oocytes are injected. This report is

in agreement with those of Liu et al. (1995). Flaherty et al., 1998, reported that the risk of

failed fertilization reduced from 37% when one oocyte was injected to only 0.8% when five

or more oocytes were injected.

The participation of sperm decondensation, meiosis resumption, oocyte activation and

pronuclei migration can be easily studied using techniques like immunofluorescence when

failed or poor fertilization occurs. It can be concluded that this techniques can be used as a

tests to predict future results in subsequent cycles.

References

Asch R., Simerly C., Ord T., Ord V.A. and Schatten G. (1995). The stages at which human fertilization
arrests: microtubule and chromosome configurations in inseminated oocytes which failed to complete
fertilization and development in humans. Mol. Hum. Reprod., 1, 1897-1906.

Dozortsev D., Rybouchkin A., DeSutter P. (1995). Human oocyte activation following intracytoplasmic
sperm injection; the role of the sperm cell. Hum. Reprod., 9: 2139-2144.

Flaherty SP., Payne D., Swann NJ., Mathews CD. (1995). Aetiology of failed and abnormal fertilization
after intracytoplasmic sperm injection. Hum. Reprod., 10: 2623-2629.

Flaherty SP., Payne D., Swann NJ., Mathews CD. (1998). Assessment of fertilization failure and abnormal
fertilization after intracytoplasmic sperm injection. Hum. Reprod. Supplement, 13:155-164.

Liu J., Nagy Z., Joris H. y col.(1995). Analysis of 76 total fertilization failure cycles out of 2732
intracytoplasmic sperm injection cycles. Hum Reprod., 10: 2630-2636.

Lopes S., Juriscova A., Casper R. (1998). Gamete- specific DNA fragmentation in unfertilized human
oocytes after intracytoplasmic sperm injection. Hum. Reprod., 13: 703-708.

Plachot, M. and Crozet, N. (1992). Fertilization abnormalities in human in- vitro fertilization. Hum. Reprod.,
7, 89-94.

Sakkas D., Urner F., Bianchi PG et al. (1996). Sperm chromatin anomalies can influence decondensation
after intarcytoplasmic sperm injection. Hum. Reprod., 11: 837-843.

Schatten G. (1994). The centrosome and its mode of inheritance: the reduction of the centrosome during
gametogenesis and its restoration during fertilization, Dev. Biol., 165: 299-335.

Schmiady, H., Sperling, K., Kentenich, H., Stauber, M. (1986). Prematurely condensed human sperm
chromosomes after in vitro fertilization (IVF). Hum. Genet., 74: 441- 443.
2+ 2+
Sousa M., Barros A. and Tesarik J. (1996). The role of rianodine- sensitive Ca stores in the Ca
oscillation machine of human oocytes. Mol. Hum. Reprod., 2: 265-272.

Swann K. (1990). A cytosolic sperm factor stimulates repetitive calcium increases and mimics fertilization in
hamster eggs. Development, 110: 1295-1302.

Tesarik J. and Sousa M. (1994). Comparison of Ca 2+ responses in human oocytes fertilized by subzonal
insemination and by intracytoplasmic sperm injection. Fertil. Steril., 62: 1197-1204.

Tesarik J. and Testart J. (1994). Treatment of sperm injected human oocytes with Ca 2+ ionophore supports
the development of Ca 2+ oscillations. Biol. Reprod., 51: 385-391.

Tesarik J.(1998). Oocyte activation after intracytoplasmic injection of mature and immature sperm cells.
Hum. Reprod. Supppl., 13 (1): 117-127.

Tesarik, J., and Kopecny, V. (1989 a). Developmental control of the human male pronucleus by ooplasmic
factors. Hum. Reprod., 4, 962-968.

Tesarik, J., and Kopecny, V. (1989 b). Development of the human male pronucleus: ultrastructure and
timing. Gamet Res., 24, 135-149.

Zenzes, M. T. and Casper, R. F. (1992). Cytogenetics of human oocytes, zygotes and embryos after in vitro
fertilization. Hum. Genet., 88: 367- 375.