Human Reproduction vol 11 no 9 pp 1985-1989, 1996
A bridge to intracytoplasmic sperm injection — high
insemination concentrations benefit patients who have a
reduced chance of fertilization with standard in-vitro
fertilization
Dickinson B.Cowan, Meryl Santis, Tracy Keefe,
Corinne A.Hargreaves, Richard J.S.Howell and
Sheryl T.Homa*
Fertility Unit, The Portland Hospital For Women and Children,
209 Great Portland Street, London WIN 6AH, UK
'To whom correspondence should be addressed
This study was carried out to determine whether high
insemination concentrations (HIC) could improve fertilization and pregnancy rates in patients who had either
previously demonstrated poor fertilization rates in vitro
using standard protocols (Group 1) or in whom a reduced
chance of fertilization was indicated at semen assessment
prior to in-vitro fertilization (TVF) (Groups 2 and 3). Forty
nine patients were recruited for the study. Standard IVF
was carried out in 1 ml volumes using 105 spermatozoa/
ml. HIC treatment involved co-culture of spermatozoa and
oocytes in microdroplets with insemination concentrations
increased 10-50 fold higher than standard IVF. Fertilization
and pregnancy rates were compared between standard IVF
and HIC in individual patients either in consecutive cycles
(Group 1) or using sibling oocytes in the same cycle (Group
2). Group 3 patients were treated with HIC for their first
treatment cycle. HIC significantly improved the fertilization
rate compared with standard IVF for Groups 1 (59.7 ±
10.7 versus 19.6 ± 5.4% respectively) and 2 (54.9 ± 8.5
versus 34.0 ± 8.5% respectively). HIC increased the
pregnancy rate from 0% with standard IVF to 20% per
embryo transfer in Group 1 patients. A single pregnancy
derived from the transfer of HIC and IVF embryos
occurred in Group 2. The fertilization rate (47.2 ± 7.6%)
and pregnancy rate (31.3% per embryo transfer) for Group
3 patients was higher than predicted. There was no increase
in the rate of polyploidy with HIC. Provided there are
sufficient numbers of motile spermatozoa, HIC may be
offered as an initial form of treatment, thus permitting
referral of only the poorest responders for intracytoplasmic
sperm injection (ICSI).
Key words: fertilization/insemination/IVF/polyploidy
Introduction
Approximately 5% of all patients undergoing standard treatment by m-vitro fertilization fail to achieve fertilization.
This may be due to defects which include chromosomal
abnormalities, immaturity of the spermatozoa or oocyte as
well as other as yet unknown biochemical parameters (reviewed
by Homa et al., 1993). Failure of fertilization may also result
© European Society for Human Reproduction and Embryology
from atypical morphology of the gametes which leads to
defective spermatozoa-oocyte interactions involving zona
binding and penetration.
The introduction of intracytoplasmic sperm injection (ICSI)
has overcome many of these problems, leading to comparable
fertilization rates, and, in some instances superior pregnancy
rates when compared with standard in-vitro fertilization techniques (Van Steirteghem et al, 1993). However, ICSI is a new,
highly invasive procedure which has not been implemented for
sufficient time to allow follow-up studies of children bom as
a result of this technique. There are concerns about the potential
genetic, biochemical and structural defects which may result
from introducing abnormal spermatozoa or immature sperm
cells into the oocyte, or from the technique of micromanipulaOon itself (Fishel et al, 1993; De Jonge and Pierce, 1995;
Patrizio, 1995). This raises interesting issues regarding whether
ICSI should routinely be used as an alternative to IVF or if it
should currently be used only as a last resort (Baker et al,
1993; Fishel et al, 1993, De Jonge and Pierce, 1995; Patrizio,
1995). As a consequence, some patients are hesitant to embark
on ICSI treatment.
The culture of spermatozoa with oocytes in vitro optimizes
the fertilization process, where the intrinsic difficulties presented to the spermatozoa in its passage through the female
reproductive tract in search of the oocyte, are overcome by
increasing the proximity of the oocyte and spermatozoa in the
culture dish or tube. There is an accepted range of sperm
concentration for achieving maximal fertilization rates in vitro,
while a further increase in sperm concentration is associated
widi an increased incidence of polyploidy (Wolf et al, 1984;
Englert et al, 1986). However, in cases of oligozoospermia
or teratozoospermia a tenfold or more increase in the standard
range of insemination concentration can be of great benefit in
improving fertilization rates (Wolf et al, 1984; Oehninger
et al., 1988; Ord et al, 1993).
The aim of this study was two-fold. The first task was to
determine whether high insemination concentrations (HIC)
could significantly benefit patients who had previously demonstrated poor rates of fertilization during standard IVF treatment,
irrespective of sperm parameters. The second task was to
investigate couples who were considered to be at risk of reduced
fertilization using standard IVF insemination techniques due
to sperm parameters. In these cases, none of the patients were
classified as being severely oligoasthenoteratozoosperrmc, but
at least one sperm parameter lay outside the normal range
(WHO, 1992). The study involved a prospective comparison
between standard IVF and HIC using sibling oocytes in the
same cycle.
1985
DJJ.Cowan et aL
Table L Patient allocation to treatment groups
Group
Indications
Study design
No patients
1
Poor fertilization rate at IVF
12
2
At least one sperm parameter outside normal range
3
At least one sperm parameter outside normal range
Standard IVF treatment cycle followed by HIC treatment in subsequent
cycle
Sibling oocyte cohort split between standard IVF and HIC in the same
cycle
All oocytes subjected to HIC
Tubal
Endometnosis
Male factor
Anti-sperm antibodies
Idiopathic
Group
1
2
3
4
2
6
2
2
6
0
7
2
2
8
0
12
2
1
"Number of diagnoses exceeds the number of patients m each group, as in
some instances there was more than one cause for treatment
Materials and methods
Patients and groups
Allocation to three treatment study groups was carried out as described
in Table I. The mean age (Group 1, 33.8 ± 1.2; Group 2, 31.8 ±
0.7, Group 3, 32.4 ± 1.0) was similar between treatment groups. The
diagnoses of infertility are shown in Table IL A total of 49 couples
was recruited for this study. Twelve of these couples had previous
attempts at standard IVF and had demonstrated poor fertilization,
including 4 with complete failed fertilization (Group 1). In the
following treatment cycle, all oocytes collected from these patients
were subjected to HIC. In the remaining 37 cases, at least one sperm
parameter (count, motility, progression, abnormal forms) fell below
the normal range (see Semen Evaluation below). Sibling oocytes
collected in the same treatment cycle from 17 of the corresponding
female partners (Group 2), were split and cultured using either
standard IVF procedures or using HIC. In the other 20 cases (Group
3), all the oocytes collected were subjected to HIC on the first
treatment cycle.
Semen evaluation
Sperm count and motility was assessed in a counting chamber
according to World Health Organization criteria (1992). Sperm
morphology was determined at a magnification of X400 on wet
preparations. A count of more than 20X10 6 , >40% motility and
> 1 5 % normal forms were considered to be within normal limits. If
the great majority of spermatozoa in a semen sample were not
progressing in a rapid, linear fashion, then such poor progression was
also considered to warrant the use of HIC.
Ovarian stimulation
In all cases, the pituitary was down-regulated with intra-nasal
Buserelin (gonadotrophin hormone-releasing hormone analogue; Suprefact: Hoechst UK Ltd, Hounslow, UK) starting on day 21 of the
previous cycle. The ovaries were stimulated by ijn. injection of
human menopausal gonadotrophins (HMG) (Pergonal: Serono
Laboratories Ltd, Welwyn Garden City, UK) and/or high purity FSH
(Metrodin: Serono Laboratories Ltd, Welwyn Garden City, UK).
Transvaginal oocyte aspiration was carried out approximately 34 h
1986
20
after injection of human chononic gonadotrophin (HCG) (Profasr
Serono Laboratories Ltd).
Table IL The diagnoses of infertility in patient groups 1-3*
Diagnosis
17
Standard IVF
Oocytes were cultured in 1 ml medium under paraffin oil in the
centre well of organ culture dishes (Falcon no. 3037: Marathon
Laboratory Supplies, London, UK) (up to six oocytes per dish) with
approximately 100 000 partner's spermatozoa/ml at 37°C under 5%
CO? in air. The medium was Earle's balanced salts solution (EBSS:
Life Technologies, Ltd, Paisley, UK) containing 1 mM sodium
pyruvate, 25 mM NaHCO3, penicillin and gentamicin, supplemented
with 10% autologous serum.
High insemination concentration procedure
Oocytes were cultured individually in 100 ml droplets of medium as
described above, under 1 ml paraffin oil. Spermatozoa were added at
a concentration of l-5X10 6 /ml.
For the split study of sibling oocytes in Group 2, oocytes were
assigned for IVF or HIC at random.
Embryo transfer and luteal support
In all cases, cumulus cells were removed mechanically 18 h postlnsemination to determine formation of pronuclei. Oocytes demonstrating two or more pronuclei at this time were considered to be
fertilized. Up to three bipronucleale embryos were transferred following a further 24 h in culture. Luteal phase support was given in the
form of Cyclogest (Hoechst UK Ltd, Hounslow, UK) pessaries
vaginally for 2 weeks post-embryo transfer. A clinical pregnancy was
confirmed upon detection of a fetal heart following transvagmal
ultrasound scan 3 weeks post-embryo transfer.
Statistics
The results are described as means together with the standard errors
of the means. The statistical significance of the difference between
the means was assessed by Student's Mest
Results
Insemination concentrations for HIC varied between 1 and
5 X10 6 per ml. There was no correlation between insemination
concentration and fertilization rate (data not shown). Table HI
shows the distribution of sperm count, motility and abnormal
forms in the different groups studied. Group 1 patients were
those who had demonstrated poor fertilization rates in at least
one previous IVF attempt, while Groups 2 and 3 spermatozoa
were considered to be 'borderline' for standard FVF. Interestingly, the mean sperm counts, percentage motility and abnormal forms all fell within the normal range as defined by the
WHO (1992), whereas the distribution of values within each
parameter fell outside the normal range for every category.
This further emphasizes the large heterogeneity of semen
High insemination concentrations for IVF
Table IIL Mean and range of sperm parameters in all treatment groups
Group
1
IVF
me
2
3
CountXIO*
Progression*
Abnormal forms %
Motibty %
93 7 (11-280)
95 0(2-200)
81 5 (7-280)
45.5 (4-260)
2 4 (2-3)
2 7 (2-3 5)
2.7 (2-3.5)
3 1 (2-4)
57 0 (25-95)
52 2(37-85)
51 6(20-93)
51 1 (34-90)
53 4 (32-70)
52 3 (27-70)
52 0 (27-80)
48 8(13-75)
"Progression grades refer only to spermatozoa with forward progression and defines the overall progression m each sample 1
mostly linear, 3 = sluggish, usually erratic, 4 = weak, erratic, non-linear
very rapid, linear, 2 = rapid.
Table IV. Comparison of fertilization and pregnancy rate between high insemination concentration (HIC) and standard IVF
Group 1
Total no eggs
Total no. embryos
2pn
3pn
% 3pn of total fertilized
No. patients that failed to fertilize
Mean % fertilization rate per patient ± SEM
normal (2pn)
total (2pn + 3pn)
% pregnancy rate
per patient
per embryo transfer
Group 2
Group 3
IVF
me
IVF
HIC
fflC
73
67
103
113
130
16
1
59
4 (33 3%)
42
1
2.3
2 (16 7%)
35
4
103
7 (41 2%)
59
1
23
3 (17 6%)
55
5
83
3 (15 0%)
184 + 5 3
19 6 ± 5.4
59 0 ± 1 0 . 9 * "
59 7 ± 10 7**
31 2 ± 79
340 ± 85
47 8 ± 8 5*
54 9 ± 8 5***
448 ± 7 5
47 2 ± 7 6
0 (0/12)
0(0/8)
16 7(2/12)
20 0 (2/10)
One pregnancy derived from
transfer of two HIC embryos
+ 1 IVF embryo
25 (5/20)
31.3(5/16)
Significantly different from IVF *P < 0 05, **P < 0 02, •**/> < 0.01
pn = pronucleate
parameters in patients where fertilization rates are sub-optimal
with conventional IVF.
A comparison of fertilization and pregnancy rates between
HIC and standard IVF for all groups of patients is shown in
Table IV. In Group 1, patients who had undergone standard
IVF treatment had relatively poor fertilization rates (19.6 ±
5.4%). However, exposing the same patients' oocytes to HIC
in a subsequent treatment cycle resulted in trebling of the
fertilization rate (59.7 ± 10.7%). In only two cases did standard
IVF give superior fertilization rates to those achieved with
HIC, but in these cases, only one and two oocytes were available
for HIC, making comparisons with IVF not particularly valid.
In Group 1, four cases resulted in failed fertilization using
standard IVF (Table IV). Following HIC in the consecutive
cycle, every one of these patients achieved fertilization rates
between 60 and 100%. There were two pregnancies in the
HIC group compared with none in the IVF group.
To ensure a more accurate control for these observations, a
comparison was made between fertilization rates using standard
IVF and HIC for sibling oocytes in the same treatment cycle.
Seventeen patients were selected for this study (Group 2).
Patients were selected using semen assessment criteria where
one or more parameters were considered to be outside the
normal range. Four of these patients had been treated by IVF
at least once before, where not only did the male partners
have sub-optimal sperm parameters, but fertilization rates were
variable. Oocytes collected from each individual from Group
2 were divided into two groups and one exposed to standard
IVF while the other was exposed to HIC. Interestingly, the
fertilization rate for the standard IVF patients in this group
(34.0 ± 8.5%) was higher than the fertilization rates for the
IVF patients in Group 1 (19.6 ± 5.4%) (Table IV), although
they were still considerably lower than the general fertilization
rate quoted nationally for all IVF patients. This is no doubt
due to the fact that poor fertilization rates had already been
established in Group 1 patients before HIC was carried out.
Total fertilization rates were significantly improved with
HIC in 12 cases in Group 2 patients (Table IV). Four of these
had failed fertilization with standard IVF but succeeded with
12.5-100% fertilization with HIC. Three cases showed complete fertilization failure with both standard IVF and HIC and,
in one case, fertilization rates were identical between the two
forms of treatment (80%). In only one case did IVF give a
superior total fertilization rate to HIC (10/11 versus 8/11
respectively) There was one pregnancy in this group of
patients. Three embryos were replaced, one derived from IVF
and two from HIC treatment
Although the fertilization rates were significantly higher
with HIC compared with standard IVF in Groups 1 and 2,
the incidence of polyploidy was similar for both treatments
(Table TV). Therefore it was felt that patients who came
through for their first treatment cycle and who were considered
to have a reduced chance of successful fertilization would
benefit from HIC for all their oocytes. Using this option
(Group 3) the total fertilization rate was 47.2 ± 7.6% (Table
TV). Again the rate of polyploidy was comparable to that
1987
D.B.Cowan et al
observed for HIC in treatment Groups 1 and 2 (Table IV).
Within this group of 20 patients (Group 3), five achieved a
pregnancy (25% per treatment cycle).
Three cases in Group 3 failed to fertilize, while in an
additional case, only one triploid embryo was obtained. In
these four cases, all of the sperm counts were below 10X106/
ml and progression was weak. However, in three other cases
with similar oligoasthenozoospermic parameters, fertilization
rates ranged from 37.5 to 66.7%.
Discussion
Our results clearly show that HIC leads to superior fertilization
rates and pregnancy rates compared with standard IVF in cases
where poor fertilization rates have previously been established.
Furthermore, we also present results from the first prospective
study comparing standard IVF with HIC using sibling oocytes
in the same cycle. They similarly show that HIC is successful
in selected cases where sperm parameters are suspected of
being sub-optimal for fertilization using standard IVF. In
contrast to previous studies which have advocated the use of
HIC in conjunction with ICSI, our evidence suggests that in
certain cases, HIC may be used as a substitute to standard
IVF without necessarily resorting to ICSI.
The use of HIC is beneficial in the majority of patients who
fail to fertilize using standard IVF protocols. In this study, a
total of 49 HIC treatments and 29 standard IVF treatments
were carried out. Failure of fertilization per patient was reduced
from 38 to 16% when HIC was used as an alternative. In a
retrospective study of patients with severe oligoasthenozoospermia, and who were treated by HIC (Ord et al., 1993)
failed fertilization was observed in 20% of cases, hi addition,
out of 28 patients who had previous failed fertilization using
standard IVF, 25 achieved fertilization with HIC in a subsequent
cycle (Ord et al., 1993). Since poor fertilization rates in one
IVF treatment cycle are not necessarily repeated in another,
the only reasonable control is to compare HIC with IVF for
such couples in the same cycle. In the present study, we have
not only confirmed the findings of Ord et al. comparing HIC
and IVF results between consecutive cycles in the same patient
but, more importantly, it was demonstrated that in the fully
controlled group using sibling oocytes, four out of seven
patients that failed to fertilize with standard IVF obtained
fertilization with HIC in the same treatment cycle.
More recent studies have compared fertilization and pregnancy rates between ICSI and HIC. In cases where patients
have previously failed to achieve fertilization using standard
IVF techniques, or where there is severe male factor infertility,
good fertilization and pregnancy rates were obtained using
HIC in microdroplets (Tucker et al, 1993). While the HIC
results appeared more convincing than those with ICSI, there
was no report of any controls being carried out in this study.
On the other hand, Hall et al. (1995) reported a sibling oocyte
control study comparing results of HIC to ICSI in cases
where normal morphology of the partner's spermatozoa ranged
between 0.5 and 4.5%. Although the fertilization rates were
marginally lower for HIC, the embryo quality, implantation
and pregnancy rates were not compromised by the procedure.
1988
The study by Hall et al. (1995) evaluated the effect of
using lXlO^/ml spermatozoa concentration; however, our
investigation did not reveal an improvement in fertilization
rate with increasing insemination concentration.
One of the dilemmas facing assisted conception units is in
regard to the selection criteria for determining which form of
treatment to offer patients for optimum results with minimum
cost and unnecessary invasive procedure. Many studies have
been published which show that new and improved technology
for assessing sperm function can be a more valid predictor of
fertilization rates in vitro than conventional semen parameters
such as a simple count and percent motility (Kruger et al,
1987; Liu and Baker, 1992; Aitken et al, 1995; Sukcharoen
et al, 1995). Tests include the ability to undergo the acrosome
reaction, the hamster egg zona binding assay, determination
of nuclear normality and computer assisted analyses of sperm
motility. More recently, it has been shown that there is an
inverse relationship between fertilization and the degree of
reactive oxygen species generated (Suckcharoen et al, 1995).
The most useful predictor for fertilization remains that of
determining sperm morphology according to strict criteria
(Kruger et al, 1987; Grow et al, 1994; Ombelet et al, 1994;
Suckcharoen et al., 1995). While fertilization and pregnancy
rates for spermatozoa which contain between 5 and 20%
normal forms can be restored to levels comparable to those
obtained for >20% normal forms using HIC, spermatozoa
which have < 5 % normal forms or counts <5X10 6 /ml may
have considerably reduced fertilization and/or pregnancy rates
(Oehninger et al., 1988; Baker et al, 1993; Grow et al, 1994;
Ombelet et al, 1994) even when HIC is employed. However,
these studies were inappropriately controlled. All patients were
categorized according to degree of teratozoospermia ranging
from normal to severe cases, yet only one study actually
examined the effects of different insemination concentrations
in each category of teratozoospermia (Oehninger et al, 1988).
However, while they controlled for sperm count and motility,
different patients were used for each insemination concentration. In only four cases were the same patients compared for
standard IVF versus HIC and these were in consecutive cycles.
In a subsequent prospective study (Ombelet et al, 1994) and
two retrospective studies (Baker ef al, 1993; Grow etal, 1994),
insemination concentrations were predetermined according to
the morphology of the spermatozoa where higher insemination
concentrations were used for increasingly higher incidences
of teratozoospermia, again no comparisons being made between
the efficacy of using HIC versus standard IVF insemination
concentration for the same individual.
While it is unclear how Baker et al. (1993) cultured
spermatozoa and oocytes, the other studies reported using HIC
in volumes that ranged between 1 and 3 ml. In contrast to the
reduced fertilization and pregnancy rates observed in these
studies, Hall et al. (1995) found that co-culturing severely
teratozoospermic (<5% normal forms) spermatozoa with
oocytes in microdroplets resulted in ~60% fertilization rates
and 22% pregnancy rates per cycle. One explanation for the
discrepancy in these results may be attributed to an alteration
in the spermatozoa to egg ratio. The reduction of actual
numbers of spermatozoa exposed to the oocyte in microdroplets
High insemination concentrations for IVF
while keeping the overall concentration of spermatozoa similar
to that used in larger volume culture, may reduce the potential
harmful effects produced by large numbers of spermatozoa in
overnight culture, such as the release of free radicals and
proteases etc. from dying spermatozoa Additionally, an alteration in the concentration of soluble secretory products released
by the cumulus cells depending on culture volume may affect
fertilization.
Although the current study has also reported successful
fertilization and pregnancy rates using HIC of spermatozoa in
microdroplet co-culture with oocytes, the majority of the
spermatozoa in this study did not fall within the category of
either severe teratozoospermia, oligozoospermia or asthenozoospermia, or combinations thereof. Yet within the study
group, a large number of patients demonstrated poor or failed
fertilization with standard IVF. Even in studies where sperm
function tests have been carried out, there are always cases
when fertilization rates have been either unpredictably low or
high. This supports the idea that while assessment of sperm
parameters and sperm function provide useful guidelines for
determining whether micromanipulation should be used, they
cannot be the only selection criteria for predicting fertilization
performance. With all the modem techniques currently available, they remain only predictors of fertilization, as neither
a single test nor any combination thereof can absolutely
predetermine the outcome, unless of course the semen is totally
azoospermic, globozoospermic or asthenozoospermic. At the
present time, the ultimate test is still the ability of the
spermatozoa of a particular individual to fertilize his partner's
oocytes in vitro. HIC does not share the potential hazards of
the ICSI technique discussed previously (Fishel et al, 1993;
De Jonge and Pierce, 1995; Patrizio, 1995). Furthermore, this
study and others have shown no significant increase in the
rate of polyploidy using HIC with spermatozoa which fall
outside the normal range (Oehninger et al., 1988; Hammitt,
1993; Ord et al, 1993; Grow et al., 1994; Hall et al., 1995).
More importantly, pregnancy rates for HIC are comparable to
those of ICSI (Tucker et al., 1993; Hall et al, 1995). For all
these reasons, in agreement with others (Baker et al., 1993;
Tucker et al., 1993; Hall et al, 1995) we therefore advocate
the use of HIC, if there are sufficient spermatozoa, as a
necessary test when predictive values are low and where
resorting to ICSI may be unnecessary.
Hall, J., Fishel, S , Green, S et aL (1995) Intracytoplasmic sperm injection
versus high insemination concentration in-vitro fertilization in cases of very
severe teratozoospermoa. Hum. Reprod., 10, 493-496
Hamrmt, D (1993) Treatment of male factor infertility by in vitro insemination
with high concentrations of motile sperm Semin. Reprod. EndocnrtoL, 11,
72-82,
Homa, S.T, Carroll, J and Swann, K. (1993) The role of calcium in
mammalian oocyte maturation and egg activation Hum. Reprod., 8,
1274-1281
Kruger, T F , Acosta, A., Simmons, K.F et aL (1987) New method of
evaluating sperm morphology with predictive value for human ln-vitro
fertilization Urology, 30, 248-251
Liu, D.Y. and Baker, H W G . (1992) Tests of human sperm function and
fertilization in vitro. FemL SteriL, 58, 465-^83.
Oehninger, S , Acosta, A A., Morshedi, M. et aL (1988) Corrective measures
and pregnancy outcome in in vitro fertilization in patients with severe
sperm morphology abnormalities. FemL StenL, 50, 283—287
Ombelet, W, Foune, F.le R., Vandeput, H et aL (1994) Teratozoospermia
and ln-vitro fertilization, a randomised prospective study. Hum. Reprod., 9,
1479-1484
Ord, T, Patnzio, P., Balmaceda, J P and Asch, R H (1993) Can severe male
factor infertility be treated without micromanipulation? FemL SteriL, 60,
110-115.
Patnzio, P (1995) Intracytoplasnuc sperm injection (ICSI) potential genetic
concerns Hum. Reprod., 10, 2520-2523
Suckcharoen, N , Keith, J , Irvine, D S and Aitken, RJ (1995) Predicting
the fertilizing potential of human sperm suspensions in vitro importance
of sperm morphology and leukocyte contamination FemL Stenl, 63,
1293-1300
Tucker, M., Wiker, S and Massey, J (1993) Rational approach to assisted
fertilization Hum. Reprod, 8, 1778
Van Steuteghem, A C , Nagy, Z , Jons, H et al (1993) High fertilization and
implantation rates after intracytoplasmic sperm injection Hum. Reprod.,
10, 1061-1066.
Wolf, D P , Byrd, W., Dandeker, P and Quigley, MM (1984) Sperm
concentration and the fertilization of human eggs in vitro BioL Reprod.,
31, 837-848
World Health Organization (1993) Laboratory Manual for the Examination of
Human Semen and Semen-Cervical Mucus Interaction, 3rd edn Cambridge
University Press, New York, pp. 1-107
Received on April 25. 1996, accepted on July 10, 1996
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