Aspiration of oocytes for in-vitro fertilization.
ABSTRACT An aspiration system, incorporating a regulated vacuum pump, was used to examine, in vitro, some factors that may affect oocyte collection. In an open aspiration system, as the length of the needle was increased, or the internal diameter decreased, the velocity (and flow rate) of aspirated fluid decreased. There was a difference, however, between experimental flows and those predicted by Hagen-Poiseuille's Law. Upon application of vacuum to a closed aspiration system, employing isolated bovine ovaries, there was an initial rapid increase in the collection tube vacuum to 85% of the selected pump vacuum followed by a more gradual rise to 100%. The vacuum within the needle similarly rose rapidly to approximately half the selected vacuum, while the vacuum at the needle tip was approximately 5% of selected vacuum. The vacuums throughout the system briefly equilibrated as maximum flow/velocity was reached. Flow/velocity slowed dramatically as the follicle collapsed, and stopped as the needle tip was blocked. If vacuum was maintained during the withdrawal of the needle from the follicle, there was a dramatic forward flow of fluid toward the collection tube. The morphological appearance of bovine cumulus after in-vitro aspiration was generally unaltered by vacuums commonly utilized in oocyte collection, providing the cumulus was regular, compact and refractile. The cumulus was less resistant to aspiration if it was damaged or had degenerated. These results suggest that an intact cumulus may offer protection during oocyte collection.
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ABSTRACT: Oocytes of nonhuman primates such as rhesus monkeys are excellent models for diverse studies on developmental biology, epigenetics, human reproduction, and assisted reproductive technologies, as well as on transgenics. Such studies require numerous oocytes that can be retrieved after controlled ovarian stimulation. Currently, most primate centers use laparoscopic aspiration or laparotomy followed by aspiration to collect rhesus oocytes, although the ultrasound-guided needle aspiration is more advantageous due to reduced infection risk, less injury, and a shorter recovery period. Yet, some initial difficulties associated with the ultrasound-guided needle aspiration limit its broader application. The objective of the present study was to address these obstacles. By presenting practical solutions to the initial difficulties, results from our study show that it is possible to collect a mean number of 38 +/- 10 rhesus oocytes per hormonally stimulated female. These results compare favorably to the average number of rhesus oocytes collected using the laparoscopic approach and suggest that when initial obstacles are overcome, the ultrasound-guided oocyte retrieval represents a good alternative to more invasive approaches.Molecular Reproduction and Development 07/2009; 76(9):890-6. · 2.81 Impact Factor
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ABSTRACT: Most studies on granulosa cell (GC) function in cattle have been performed using GC and follicular fluid (FF) samples collected from slaughterhouse ovaries. Using this approach, the follicular developmental stage and functional status are unknown and indirectly inferred, limiting data interpretation. Ultrasound-guided follicle aspiration has previously been used to recover GC or FF samples, but this was mostly carried out in large follicles or pools of small follicles, without recording the efficiency of recovery. The present study was aimed at adapting and evaluating an ovum pick-up (OPU) system for the in vivo recovery of FF and GC from individual follicles of different diameters. In the first trial, the losses of fluid inside the tubing system were calculated using a conventional or an adapted-OPU system. Blood plasma volumes equivalent to the amount of FF in follicles of different diameters were aspirated using a conventional OPU Teflon circuit. The OPU system was then adapted by connecting 0.25 mL straws to the circuit. A second trial evaluated the efficiency of FF recovery in vivo. Follicles ranging from 4.0 to 16.8 mm in diameter were aspirated individually using the conventional or adapted-OPU systems. A third trial assessed the in vivo recovery of GC and the subsequent amount of RNA obtained from the follicles of different diameters from Holstein and Gir cattle. In Trial I, the plasma recovery efficiency was similar (P > 0.05) for the volumes expected for 12 and 10 mm follicles, but decreased (P < 0.05) for smaller follicles (45.7+/-4.0%, 12.4+/-4.3% and 0.0+/-0.0% for 8, 6, and 4 mm follicles, respectively). Using the adaptation, the losses intrinsic to the aspiration system were similar for all follicle diameters. In Trial II, the expected and recovered volumes of FF were correlated (r = 0.89) and the efficiency of recovery was similar among follicles <12 mm, while larger follicles had a progressive increase in FF losses that was not related to the tubing system. In Trial III, the number of GC and amount of RNA obtained were not affected (P > 0.05) by follicle size, but differed according to breed (615,054+/-58,122 vs 458,095+/-36,407 for Holstein and Gir, respectively; P < 0.05). The adapted-OPU system can be successfully used for the in vivo collection of FF and GC from follicles of different diameters. This will enable further endocrine, cellular, and gene expression analyses.Reproductive Biology and Endocrinology 01/2013; 11(1):73. · 2.14 Impact Factor
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ABSTRACT: The objective of this study was to determine whether or not reproductive performance in cattle produced by somatic cell nuclear transfer (SCNT) is significantly different from that of their genetic donors. To address this question, we directed two longitudinal studies using different embryo production procedures: (1) superovulation followed by artificial insemination (AI) and embryo collection and (2) ultrasound-guided ovum pick-up followed by in vitro fertilization (OPU-IVF). Collectively, these two studies represent the largest data set available for any species on the reproductive performance of female clones and their genetic donors as measured by their embryo production outcomes in commercial embryo production program. The large-scale study described herein was conducted over a six-year period of time and provides a unique comparison of 96 clones to the 40 corresponding genetic donors. To our knowledge, this is the first longitudinal study on the reproductive performance of cattle clones using OPU-IVF. With nearly 2,000 reproductive procedures performed and more than 9,200 transferable embryos produced, our observations show that the reproductive performance of cattle produced by SCNT is not different compared to their genetic donors for the production of transferable embryos after either AI followed by embryo collection (P = 0.77) or OPU-IVF (P = 0.97). These data are in agreement with previous reports showing that the reproductive capabilities of cloned cattle are equal to that of conventionally produced cattle. In conclusion, results of this longitudinal study once again demonstrate that cloning technology, in combination with superovulation, AI and embryo collection or OPU-IVF, provides a valuable tool for faster dissemination of superior maternal genetics.PLoS ONE 01/2013; 8(12):e84283. · 3.53 Impact Factor
Human Reproduction Update 1996, Vol. 2, No. 1 pp. 77–85
European Society for Human Reproduction and Embryology
Aspiration of oocytes for in-vitro fertilization
Robert Horne1, Christopher J.Bishop1,3, Geoff Reeves1, Carl Wood2 and
1Cook Australia, 12 Electronics Street, Eight Mile Plains Q 4113 and 2Monash IVF, 181 Hoddle Street, Richmond, Victoria
TABLE OF CONTENTS
Materials and Methods
An aspiration system, incorporating a regulated vac-
uum pump, was used to examine, in vitro, some factors
that may affect oocyte collection. In an open aspiration
system, as the length of the needle was increased, or the
internal diameter decreased, the velocity (and flow
rate) of aspirated fluid decreased. There was a differ-
ence, however, between experimental flows and those
predicted by Hagen–Poiseuille’s Law. Upon applica-
tion of vacuum to a closed aspiration system, employ-
ing isolated bovine ovaries, there was an initial rapid
increase in the collection tube vacuum to 85% of the
selected pump vacuum followed by a more gradual
rise to 100%. The vacuum within the needle similarly
rose rapidly to approximately half the selected vac-
uum, while the vacuum at the needle tip was ∼5% of
selected vacuum. The vacuums throughout the system
briefly equilibrated as maximum flow/velocity was
reached. Flow/velocity slowed dramatically as the fol-
licle collapsed, and stopped as the needle tip was
blocked. If vacuum was maintained during the with-
drawal of the needle from the follicle, there was a dra-
matic forward flow of fluid toward the collection tube.
The morphological appearance of bovine cumulus
after in-vitro aspiration was generally unaltered by
vacuums commonly utilized in oocyte collection, pro-
viding the cumulus was regular, compact and refrac-
tile. The cumulus was less resistant to aspiration if it
was damaged or had degenerated. These results
suggest that an intact cumulus may offer protection
during oocyte collection.
Key words: damage/in-vitro fertilization/oocyte
The initial studies on the maturation of human oocytes in
vitro were carried out on oocytes which were obtained
when ovaries or pieces of ovaries were acquired by laparo-
tomy (Edwards, 1965). By 1970, Steptoe and Edwards had
developed a laparoscopic method for aspirating oocytes
from their Graafian follicles, with a method that yielded
oocytes from approximately one-third of follicles. Initially,
they used a needle and syringe to provide the suction, but
later developed an aspiration device which provided con-
tinuous suction, with control being exerted by the assist-
ant’s finger on the bypass valve. The vacuum used did not
usually exceed 120 mm Hg, in order to avoid damage to the
oocyte (Edwards et al., 1980). A similar technique, using a
Venturi system at 200 mm Hg activated by a foot-operated
‘on–off’ valve, was utilized by Lopata et al. (1974).
Following the first few births from in-vitro fertilization
(IVF) and embryo transfer (Steptoe and Edwards, 1978),
attention was focused on the instruments used for oocyte
recovery. Equipment, including a variety of needles and
regulated aspiration pumps, became commercially avail-
able in the early 1980s; for example, a ‘Teflon’ coated needle
was developed that resulted in oocyte collection rates of
>90% (Renou et al., 1981). The next major development
was the change from laparoscopic to transvaginal ultra-
sound-guided aspiration (Feichtinger and Kemeter, 1986).
Apart from a comparison of manual and mechanical
suction on the effect of zonal damage (Cohen et al., 1986),
there has been surprisingly little published on the theory of
oocyte collection. In this article, we report the investiga-
tions of a number of factors that may affect oocyte collec-
tion and/or damage the ova. These include variables such
as pump vacuum, velocity, needle lumen size and length,
follicular pressure and size, and collection techniques.
3To whom correspondence should be addressed
78 R.Horne et al.
Figure 1. Diagrammatic representation of aspiration system.
Materials and methods
The aspiration system, shown in Figure 1, incorporated a
regulated vacuum pump (K-MAR-5000; Cook Medical
Technology, Brisbane, Australia) to which was connected,
in line, a fluid shut-off valve (Vacu-gard, Abbott, USA),
2.75 m of PVC tubing with an internal diameter (i.d.) of
5.0 mm, either a 15 ml or a 50 ml collection tube (Falcon
Plastics, Lincoln Park, NJ, USA) and a 50 cm long Teflon
line (1.2 mm i.d.) attached to a 50 cm long stainless steel
16-gauge needle (1.2 mm i.d.). A clamp was utilized to
maintain the collection tube at a known height.
Pressure transducers were connected, via 0.4 mm i.d.
side-arms, at various points in the system. The side-arms
were located in the collection tube (P3), and at the needle/
line intersection (P4). In the initial characterization of the
system, the needle tip was inserted into a large open fluid
reservoir, rather than the follicle described in the figure. In
this configuration, the fluid pressure was measured within
the reservoir, just external to the needle tip, utilizing the
needle tip transducer (P5). In experiments utilizing bovine
follicles, the pressure inside the follicle being aspirated was
recorded either via the transducer attached to the needle tip
(P5) or via a separate 22-gauge needle attached to the same
transducer. In some experiments, various characteristics of
the system were changed and the relative time taken for the
vacuum in the collection tube (P3) to match the selected
vacuum (P2) was noted.
Signals from these transducers were transmitted to an ana-
logue-to-digital converter within a PC AT computer, allow-
ing the collection of 10 measurements per second from each
transducer with an accuracy of ±0.3 kPa (2.3 mm Hg).
Determination of velocity and flow rate
Velocity and flow rate through the needle and attached
lines were calculated from the pressure difference between
the collection tube (via transducer P3) and the needle tip
(via transducer P5). Accordingly, the velocity and flow rate
were slightly underestimated, especially at the moment the
needle punctured the wall of the follicle.
The calculation of velocity of the fluid within the needle,
for a given pressure, used the following model:
av2 + bv + c = 0
where v is the velocity of the fluid through the aspiration
system and a represents losses at changes of cross-section,
2?1 ? K2?A2
and b represents frictional resistance of the needle and line,
and c is the pressure and gravitational driving force, ex-
c ? (P3? P5) ? ?g(z3? z5)
This model incorporates Hagen–Poiseuille’s Law ac-
counting for shear stress and describes a non-linear rela-
tionship between velocity and pressure. The symbols used
in the above equations are as follows: ρ = density of the
fluid, µ = viscosity of the fluid, g = gravitational acceler-
ation (= 9.81 m/s2), K1 = loss factor for the inlet to the
needle, K2 = loss factor for the needle/line interface, A1 =
cross-sectional area of the needle, A2 = cross-sectional area
of the aspiration line, L1 = length of the needle, L2 = length
of the aspiration line, D1 = diameter of the needle, D2 =
diameter of the aspiration line, P3 = pressure at the collec-
tion tube, P5 = pressure at the follicle, z3 = vertical distance
at the collection tube from a datum, z5 = vertical distance at
the follicle from a datum.
Aspiration of oocytes 79
Flow rate, Q, was calculated by Q = vA, where v is the
velocity of the fluid and A is the internal cross-sectional
area of the needle or line.
Empirical data were collected using the aspiration sys-
tem to test the validity of this model.
Pressure changes in bovine follicles
The ovaries were supplied stored in sealed bags on ice by a
local abattoir and were used within 6 h of collection. The
follicles were graded to approximate sizes of 7, 10 and
15 mm in diameter before being aspirated using the above
aspiration system at a variety of vacuums ranging from 2.5
(19 mm Hg) to 35 kPa (262.5 mm Hg).
Pressures and flow rates obtained using bovine follicles
were compared against measurements obtained using a
simulated ‘follicle’ consisting of the thumb of a latex surgi-
cal glove, securely attached to the end of the needle. The
‘follicle’ was filled with fluid such that it attained a size and
internal pressure similar to a large bovine follicle (∼15 mm
diameter and 4–8 mm Hg pressure) before being aspirated
with vacuums ranging from 5 (37.5 mm Hg) to 35 kPa
(262.5 mm Hg).
Damage to bovine cumulus mass
Bovine ovaries were collected as described above. The
oocytes were aspirated, as described above, from the fol-
licles at regulated vacuums of 5–10 kPa (37.5–75 mm Hg).
The oocytes were then transferred by pipette through two
changes of human tubal fluid (HTF) medium to remove as
much follicular fluid as possible.
The morphology of the cumulus mass surrounding each
oocyte was classified in three categories according to its
appearance: 1, compact mass of refractile cells; 2, loose
mass of shrunken, dense cells; 3, compact mass of pale,
Groups of three oocytes with the same follicular and
morphological classification were sorted into 15 ml Falcon
tubes with 2–2.5 ml of HTF medium. A vacuum was
chosen and the contents of the Falcon tube were aspirated
to another collection tube using the aspiration apparatus
described above but with a 17-gauge needle (1.0 mm i.d.)
in order to maximize shear stress. The oocytes were then
checked under the microscope for changes to the appear-
ance of their cumulus mass. Their appearance was scored
using the following categories: A, no change to cumulus
mass; B, lost some of outer cumulus mass cells; C, denuded
but with corona radiata intact; D, totally denuded; E,
The aspiration procedure was repeated at a higher vac-
uum and the appearance of the cumulus mass scored again.
Figure 2. Typical pressure changes within the aspiration system
where regulated vacuum is set at 15 kPa (112.5 mm Hg).
Vacuum and flow profiles in open aspiration system
Figure 2 shows the pressure changes within an open aspir-
ation system with a regulated vacuum of 15 kPa (112.5 mm
Hg) attached to a reservoir. On activation of the foot pedal,
there was an initial rapid increase in the vacuum present in
the collection tube (P3) followed by a more gradual rise to
the set vacuum. The vacuum within the needle (P4) simi-
larly rose rapidly to approximately half the set vacuum.
The vacuum decreased from the pump to the needle tip
such that the vacuum at needle tip in the aspiration system
described above was ∼5% of the selected pump vacuum
The vacuum within the collection tube (P3) reached 85%
of the selected vacuum (i.e. 85% of the regulated vacuum
in the pump) within 0.5 s. Subsequently, it took approxi-
mately a further 5 s to reach the selected vacuum. Changes
to the aspiration system did not significantly decrease the
time taken to reach 85% of the selected vacuum (not
shown), but decreased the time taken to reach 100% of the
selected vacuum (Table II).
Table I. Approximate relative vacuums (%) within aspiration
system at certain times after activation
Relative vacuum at site
(P2)(P3) (P4) (P5)
After 1 s
100 85 505
After 5 s 100100576
80 R.Horne et al.
Figure 3. Comparison of Hagen–Poiseuille’s law and experimental
and model flow rates.
Table II. Effect of changes to initial aspiration system on time
taken for vacuum in collection tube to rise from 0% to 100% of
the selected vacuum
Change to aspiration systemDecrease in
Shorten length of vacuum line from 2.75 to 0.25 m20
Decrease i.d. of vacuum line from 5 to 3.2 mm25
Use 15 ml collection tube rather than 50 ml tube15
Double size of vacuum reservoir in pump20
Changing all of above70
Placing valve before collection tube90
Flow predictions using the mathematical model
Figure 3 shows the difference between experimental flows
and those predicted according to Hagen–Poiseuille’s Law.
It indicates that the Hagen–Poiseuille Law for steady flow
through pipes does not accurately model the system. Figure
3 also shows the calculated flows according to the math-
ematical model described above. While the flow remains
laminar, the flow rates predicted by the model are within
±5% of the observed flows. In the needles tested, i.d.
<1.4 mm, laminar flow occurred over the range of vac-
uums 5–40 kPa (37.5–300 mm Hg). However, in a
16-gauge needle (1.2 mm i.d.) flow begins to become non-
laminar at vacuums >50 kPa (375 mm Hg), and the model
predicts velocities (and hence flow rates) in excess of those
Flow rate within aspiration system
The flow rates calculated for the system depicted in Figure
2 are shown in Figure 4: an initial rapid increase in flow
occurs within the needle, followed by a more gradual rise to
the maximum flow, with a similar profile to the vacuum
curve in the collection tube.
Figure 4. Typical flow rate within the aspiration system where regu-
lated vacuum is set at 15 kPa (112.5 mm Hg).
Figure 5. Relationship between length of a 16-gauge needle (1.2 mm
i.d.) and velocity at various vacuums.
Relationship of needle length and diameter to velocity
Figure 5 shows the effect of increasing needle length, using
a 16-gauge (1.2 mm i.d.) needle, with a 60 cm Teflon line
attached, on velocity (and hence flow rate) at various vac-
uums. As the length of the needle was increased, the vel-
ocity, and flow rate, decreased.
Figure 6 shows the effect of decreasing the i.d. of the
needle lumen (50 cm needle with 50 cm line) on velocity at
various vacuums. As the i.d. was decreased, the velocity
Relationship between follicular diameter, follicular
volume and internal needle volume
Figure 7 shows the relationship between follicular diam-
eter, follicular volume and the internal volume of a typical
16-gauge and a 17-gauge needle and line (needle and line
i.d. of 1.2 mm and 1.0 mm respectively; total needle and
line length of 100 cm). For example, a follicle with a diam-
eter of 13 mm has a fluid volume of ∼1.2 ml, the contents of
which would fill the lumen of a 16-gauge needle and line
(100 cm total length).
Aspiration of oocytes 81
Figure 6. Relationship between i.d. of needle and velocity at various
Figure 7. Relationship between diameter of follicle and volume of
follicle. Arrows indicate the internal volume of standard 16-gauge
and 17-gauge needles and lines (100 cm length).
Pressure profiles within follicles
Large bovine follicles have a small positive pressure of
0.5–1.0 kPa (3.75–7.5 mm Hg). Follicular pressure was
dependent on the size (and hence the maturity), shape and
position of the follicle, with the pressure increasing with
increasing follicular size (not shown).
As the needle tip punctured the follicle wall, the pressure in
the follicle increased, particularly if the needle was blunt. For
example, a sharp 22-gauge needle increased the pressure
∼1 kPa (7.5 mm Hg), while a blunt 17-gauge needle increased
the pressure by >8 kPa (60 mm Hg) before a puncture was
achieved. Once the tip penetrated the wall, in the absence of
an applied vacuum, the follicular fluid was forced out of the
follicle by the positive pressure, either up the needle lumen or
between the outer needle wall and the follicular wall.
Application of vacuum to a follicle
Vacuum applied after needle entry into the follicle
The changes in the vacuum occurring within the follicle and
a closed aspiration system after the insertion of the needle
and upon initiation of aspiration are shown in Figure 8. Upon
Figure 8. Pressure changes within the aspiration system (set at 15
kPa) during aspiration of a 15 mm diameter bovine follicle.
Figure 9. Flow rate changes within the aspiration system (15 kPa)
during aspiration of a 15 mm diameter bovine follicle.
application of vacuum, the vacuums throughout the system
equilibrated to the steady flow conditions, for a period of
time depending on the follicle volume, the regulated vacuum
used and the capacity of the needle (not shown). During this
time the follicle wall collapsed as the fluid volume decreased
until the follicle totally collapsed and blocked the needle tip.
Subsequently, the vacuum in the aspiration line and needle
increased to that in the collection tube.
The flows achieved within the closed aspiration system,
as described in Figure 8, are shown in Figure 9. The regions
of application of vacuum, steady state and follicular col-
lapse were the same as shown in Figure 8. Maximum flow
was achieved during the steady state then slowed dramati-
cally as the follicle collapsed, blocking the needle tip. In
some cases the fluid continued to flow very slowly up the
needle, possibly due to air being sucked into the follicle
around the point of entry of the needle (i.e. the system was
not fully closed).
Vacuum deactivated before exit from the follicle
Figures 10 and 11 show the changes in vacuum and flow if
the regulated vacuum was discontinued whilst the needle tip
was still in the follicle, providing the system remained closed
82 R.Horne et al.
Figure 10. Pressure changes within the aspiration system (15 kPa)
when aspiration is stopped before removal of the needle tip.
Figure 11. Flow rate changes within the aspiration system (15 kPa)
when aspiration is stopped before removal of the needle tip.
(i.e. there were no air leaks). After the pump was deactivated
and the pressure in the collection tube was returned to atmos-
pheric pressure, there was a back flow of fluid towards the
follicle. The magnitude of the back flow depended on
(i) how much air entered the system, and (ii) the height of the
collection tube above the needle tip (not shown).
Vacuum applied before entry into the follicle
It is shown in Figure 12 that if the vacuum was activated
prior to puncturing the follicle, the vacuums throughout the
system, including internally in the regulated pump, were
lower than if liquid was flowing through the needle. For
example, a regulated vacuum of 15 kPa (112.5 mm Hg)
equilibrated to 12 kPa (90 mm Hg), the vacuum in the
collection tube to 10.5 kPa (79 mm Hg), the vacuum in the
line to 5 kPa (37.5 mm Hg) and the vacuum at the needle tip
to 0.5 kPa (4 mm Hg). After the needle entered the follicle,
the pressure curves went through a transition from air flow
to fluid flow, and from an open to a closed system.
Vacuum deactivated after exit from the follicle
Figure 13 shows a comparison of flow rates, during aspir-
ation of a 15 mm bovine follicle, where both activation and
Figure 12. Pressure changes within the aspiration system (15 kPa) as
the needle tip punctures the bovine follicle where vacuum is already
applied to the needle.
Figure 13. Pressure changes within the aspiration system (15 kPa)
during the aspiration of a 15 mm diameter follicle where activation
and deactivation of the vacuum occurs outside of the follicle.
deactivation of the vacuum occurred outside the follicle.
Flow decreased as the follicle collapsed but there was a
sudden rapid flow, towards the collection tube, as the
needle was withdrawn from the follicle.
Damage to bovine oocytes
The flow rates and maximum velocities achieved in the
aspiration system using a 17-gauge (1.0 mm i.d.) needle at
various vacuums are shown in Table III. The effect of
aspiration on the morphology of the oocyte cumulus mass
is shown in Figures 14–16. Figure 14 shows that all cat-
egory 3 oocytes had lost their cumulus mass after aspir-
ation at 20 kPa (150 mm Hg). Category 2 oocytes (Figure
15) lost their cumulus mass between 30 and 40 kPa
(225–300 mm Hg), while some category 1 oocytes (Figure
16) did not lose their cumulus mass until they were aspir-
ated at 60 kPa (450 mm Hg).
Aspiration of oocytes 83
Figure 14. Appearance of category 3 bovine oocytes after aspiration
(A, no change; C, denuded but intact corona radiata; D, totally de-
nuded; E, oocyte destroyed).
Table III. Maximum flow rates and maximum velocities within
a 17-gauge needle
kPa (mm Hg)
40 (300) 1.0060.898
50 (375) 1.216 1.086
Vacuum profiles in aspiration system
Upon activation of the aspiration system described above,
attached to an open reservoir of fluid, there is an initial
rapid increase in the vacuum present in the collection tube
followed by a more gradual rise to the regulated vacuum.
The initial response is due to the presence of an evacuated
volume within the pump that becomes available when the
pump is activated. The later response is the slower evacu-
ation of the remaining air by the pump.
Usually, for oocyte collection, the vacuum in the collec-
tion system is <20 kPa (150 mm Hg). It is estimated that,
using 20 kPa (150 mm Hg), it will take ∼5 s for the system
described above to stabilize to the selected vacuum.
Changes to the aspiration system, such as shortening the
vacuum tube between the pump and the collection tube,
decreased the time for the system to reach the selected
vacuum (in the above example it was reduced from 5 to 4 s)
but did not significantly decrease the time taken to reach
85% of the selected vacuum (in the above example it was
reduced from 0.6 to 0.5 s). Given that the time taken to
aspirate follicles of 10–15 mm diameter is between 2 and 4
Figure 15. Appearance of category 2 bovine oocytes after aspiration
(A, no change; B, loss of some outer cumulus cells; C, denuded but
intact corona radiata; D, totally denuded; E, oocyte destroyed).
Figure 16. Appearance of category 1 bovine oocytes after aspiration
(A, no change; B, loss of some outer cumulus cells; C, denuded but
intact corona radiata; D, totally denuded).
s respectively, this decrease in time to reach 100% selected
vacuum may not significantly influence oocyte aspiration.
Pressure profiles within follicles
The positive pressure in the follicle is dependent on the size
(and hence the maturity), shape and position of the follicle.
As the follicle matures it produces fluid, coalescing in the
follicular antrum. This raises the pressure and causes the
bulging of the external surface, especially as it reaches
The pressure of the fluid in the follicle at the moment of
penetration of the needle may be much higher than the
normal follicular pressure. As the needle tip is being forced
into the wall, the deformation of the surface of the follicle
will cause the pressure to rise. The more blunt the needle,
the higher the pressure will become (up to 60 mm Hg) and
consequently, the larger the amount of fluid which spurts
out of the follicle when punctured. Some of this fluid will
flow up the needle, while the remainder escapes between
the outer needle wall and the follicular wall. If a vacuum
84 R.Horne et al.
has already been applied before the needle punctures the
follicle, little follicular fluid is lost.
Follicular and needle volumes
With an increased interest in oocyte collection from imma-
ture follicles, it is important to note that the volume of such
follicles is small. For example, an immature follicle with a
diameter of 5 mm has a volume of ∼0.065 ml. It would take
the contents of over 17 such follicles to fill the lumen of a
standard 16-gauge needle and line (total length 100 cm).
Application of vacuum
Once a needle punctures the follicle, the pressure within the
follicle and needle equilibrates. The follicular wall will
generally make a tentative seal around the needle and, in
the absence of an applied vacuum, resistance within the
needle will bring the fluid to rest. As the regulated vacuum
is applied, the vacuums throughout the system tend to
equilibrate to steady flow conditions. These conditions
prevail for a period of time that is dependent on the follicle
volume, the regulated vacuum used and the capacity of the
needle. For small follicles, this stage may not be reached.
As the follicular fluid volume decreases, the follicle col-
lapses. Providing a good seal exists around the needle tip,
the follicle collapses totally and blocks the tip of the needle,
causing the vacuum in the aspiration line and needle (P4
and P5) to rise rapidly, approaching the vacuum in the
collection tube (P3). The flow that was established before
the follicle wall began to collapse now begins to slow dra-
matically until the fluid stops when the needle tip is com-
In the above situation, where a good seal exists around
the needle tip, if the regulated vacuum is discontinued
whilst the needle tip is still in the follicle, there is a back
flow of fluid toward the follicle. In this closed system the
magnitude of the reverse flow is similar to the maximum
flow toward the collection tube but only lasts for a fraction
of a second and slows rapidly. This reverse flow is due to
the presence of a high vacuum in the follicle relative to the
collection tube, which has reverted to atmospheric pressure
as the pump is deactivated.
If the needle is withdrawn from the follicle while the
vacuum is still applied, there is a dramatic surge of fluid
toward the collection tube. The needle tip goes from the
high vacuum of the follicle to atmospheric pressure. This,
combined with the change from aspirating air instead of
liquid, causes the flow rate to increase rapidly. As the air
comes closer to the collection tube along the aspiration
line, the vacuum gradient increases and the fluid flow in-
creases further. If the oocyte is contained in the last fraction
of the collected follicular fluid, or comes from an immature
follicle where the volume is small, it can be subjected to
speeds well above those expected. It can also be subjected
to increased turbulence in both the needle and collection
Effect of aspiration on bovine oocyte morphology
During the collection process oocytes may be damaged by
vacuum, velocity, and/or turbulence. This may occur with-
in the needle/vacuum lines or within the follicle itself.
Damage within the needle/vacuum lines
There is a vacuum gradient down the collection system,
with the vacuum at the needle tip being only 5% of the
vacuum selected at the vacuum pump. The ovum is there-
fore exposed to an ever increasing vacuum during its travel
along the collection system. This increased vacuum may
cause the ovum to swell and the zona to crack.
High velocities may strip the cumulus of the oocyte.
Even in laminar flow, there will be a significant difference
between the velocity of the fluid in the centre of the needle
and that towards the periphery. Thus the outer layers of the
cumulus may be subjected to ‘drag’, which may strip them.
It should be remembered that, providing the selected
pump vacuum remains constant, as the oocyte moves from
a large lumen into a smaller lumen, its velocity increases.
Also, to achieve the same flow rate through a smaller diam-
eter needle, the fluid will have an increased velocity (for
example, to obtain the same flow rate through a 17-gauge
needle as through a 16-gauge needle the velocity of the
fluid in the 17-gauge will be ∼20% higher). In addition, the
longer the needle, or the smaller its i.d., the greater the
vacuum required to maintain the same velocity and the
greater the risk of damaging the oocyte.
Finally, if turbulent flow is present the ovum may be
tossed about, which could result in either stripping off of
the cumulus or cracking of the zona.
Damage within the follicle
Providing the selected pump vacuum is constant and that
there is constant lumen size, the velocity of the fluid is the
same throughout the needle and line. Thus the ovum has to
be accelerated from a resting state within the follicle to the
velocity of the fluid within the needle. Moreover, it has to
accelerate to this velocity as it enters the needle tip. This
rapid acceleration may strip off the cumulus. In theory, this
damaging effect should be greatest in smaller follicles,
especially immature follicles, where there may be some
adherence of oocytes, necessitating the use of higher suc-
tion vacuums. Additionally, the oocyte will be drawn
closer to the needle tip as the follicle collapses. This means
Aspiration of oocytes 85
that it could be subjected to an increasing accelerative force
once it detaches from the wall. This may cause the cumulus
to tear off from the oocyte. In addition, there is a rapid
increase in vacuum at the needle tip which may also affect
Damage to the cumulus
The above results indicate that an intact cumulus may be an
important factor in the resistance of oocytes to damage.
The morphology of bovine cumulus was not changed after
in-vitro aspiration at vacuums and velocities above those
normally used in vivo, providing the cumulus was regular,
compact and refractive. The cumulus was less resistant if it
was damaged or degenerated.
The above findings highlight two important issues relat-
ing to oocyte collection. Firstly, maintenance of suction:
follicular fluid (and oocytes) may be lost if entry into and
exit from the follicle are made in the absence of suction.
This gain, however, may be offset by possible damage due
to the dramatic forward flow of fluid toward the collection
tube. Secondly, movement of the needle tip within the fol-
licle: damage to the oocyte, particularly the cumulus, may
occur because of collection technique. It is a common prac-
tice during oocyte collection to ‘spin’ the needle within the
follicle. It is possible that significant damage may occur as
the oocyte is ‘scraped’ from the follicular wall by the edge
of the needle, particularly in small follicles or in the col-
lapsed follicle, where the needle size becomes large com-
pared to the follicular volume.
There is a need to undertake further studies on the effect
of needle movement in follicles on oocyte quality and
subsequent blastocyst development. One possible solution,
however, may be to combine flushing of follicles with
lower suction vacuums.
Cohen, J., Avery, S., Campbell, S., Mason, B. A., Riddle, A. and Sharma, V.
(1986) Follicular aspiration using a syringe suction system may
damage zona pellucida. J. In Vitro Fertil. Embryo Transfer, 4,
Edwards, R.G. (1965) Maturation in vitro of human ovarian oocytes.
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Edwards, R.G., Steptoe, P.C. and Purdy, J.M. (1980) Establishing full-term
human pregnancies using cleaving embryos grown in vitro. Br. J.
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Feichtinger, W. and Kemeter, P. (1986) Transvaginal sector scan
sonography for needle guided transvaginal follicle aspiration and
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Lopata, A., Johnstone, I.W.H., Leeton, J.F., Muchnicki, D., Talbot, T.M.
and Wood, C. (1974) Collection of human oocytes at laparoscopy and
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Renou, P., Trounson, A.O., Wood, C., and Leeton, J.F. (1981) The
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Steptoe, P.C. and Edwards, R.G. (1970) Laparoscopic recovering of
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Received on March 3, 1995; accepted on November 29, 1995