ELSEVIER

COMPARISON OF ULTRASOUND-GUIDED VS LAPAROSCOPIC TRANSVAGINAL

OVUM PICK-UP (OPU) IN SIMMENTAL HEIFERS

B. Santl, 1 H. Wenigerkind,1W. Schernthaner, 1 J. M6dl, 1 M. Stojkovic,2 K. Prelle,2

W. Holtz,3 G. Brem 4 and E. Wolfl'2'a

1Bavarian Research Center for Biology of Reproduction (BFZF) and

2Department of Molecular Animal Breeding and Genetics
D-85764 Oberschleil3heim, Germany
3 . . . . ,. •
Institute for Animal Breeding and Genetics, D-37075 Gottmgen, Germany
4 . . . . . .
Institute for Animal Breeding and Genetics, A-1210 Wlen, Austria

Received for publication: December 15, 1997

Accepted: March 23, 1998

ABSTRACT

Transvaginal ovum pick-up (OPU) offers several advantages over standard embryo
transfer procedures. For a systematic comparison of the ultrasound-guided (U-OPU) and the
laparoscopic OPU (L-OPU) method, groups of Simmental heifers were subjected to 1 of the 2
OPU-methods for 8 wk (15 sessions) followed by a treatment-free interval of 11 wk and then
another 8-wk period of OPU using the alternative method. Parameters that were evaluated
included the number of follicles aspirated, number and morphology of recovered cumuius-
oocyte complexes (COCs), and developmental capacity of oocytes after in vitro maturation
(IVM) and fertilization 0VF). Blood samples were also taken from the donors twice a wk for
progesterone measurement. To evaluate effects of long-term OPU on subsequent fertility of
donors, all heifers (n=14) were inseminated during the first natural estrus after the OPU
experiment. The proportion of Class I oocytes was significantly (P<0.001) higher after U-OPU
than after L-OPU (38.7% vs 21.0%). Following IVIVl/IVF, this difference in oocyte quality was
reflected by the cleavage rate (U-OPU: 58.1%; L-OPU: 52.1%; P<0.05) and the rate of
development to morulae and blastocysts (U-OPU: 27.1%; L-OPU: 13.9%; P<0.001). Among
other factors, the greater changes in vacuum pressure during L-OPU vs U-OPU might be
responsible for the difference in oocyte quality. This problem may be overcome by a more
flexible system for regulating the vacuum. Progesterone levels were higher during the L-OPU
than the U-OPU periods. Seven donors (50%) were diagnosed pregnant by ultrasonography on
Day 28 and by palpation per rectum on Day 42.
1998 by ElsevierScience Inc.
Key words: OPU, cattle, laparoscopy, ultrasonography

Acknowledgments
This study was supported by a grant from the Bayerische Forschungsstiftung (76/93).
aCorrespondence and reprint requests: Eekhard Wolf, BFZF, HackerstraBe 27, D-85764
OberschleiBheim, Germany (ewolf@lmb.uni-muencben.de).

Theriogenology50:89-100, 1998 0093-691X/98/$19.00

© 1998 by ElsevierScience Inc. PII S0093-691X(98)00116-2
90 Theriogenology

INTRODUCTION

Transvaginal ovum pick-up provides several advantages over standard embryo transfer
procedures. Since donors are usually not stimulated, the cost of hormones is eliminated.
Harvesting can be conducted twice a wk for several months, and up to the first 3 mo of
pregnancy in lactating cows (12,22). The repeated recovery of oocytes permits production of
more embryos than might be possible by standard embryo transfer practices (9). Furthermore, in
vitro fertilization of recovered oocytes allows the use of different sires for individual donors (8).
Ultrasound-guided follicle aspiration (U-OPU) was originally established by Pieterse et al.
(17,18), and is the most widely used method for OPU in cattle (7). Reichenbach et al. (19,20,21)
developed a laparoscopic procedure (L-OPU) for transvaginal follicle aspiration and discussed a
number of potential advantages of their method over U-OPU. These include 1) the aspiration of
primarily superficial follicles, 2) the direct view at the ovary and the aspiration procedure, and 3)
a reduced risk of injury to the ovary.
To our knowledge there is only one study in which a comparison was made of the efficacy
of U-OPU vs L-OPU (1). However, in this study both methods were used for different animals
and for only a limited number of OPU sessions (3 to 7 per donor), with different follicle
aspiration systems for both methods (differences in needle diameter and length as well as in the
flow rate of the medium).
To assure a systematic comparison of these methods, we set up an experiment in which
groups of Simmental heifers were subjected to U-OPU or L-OPU for 8 wk (15 sessions)
followed by a treatment-free interval of 11 wk and another 8-wk period of OPU using the
alternative method. The evaluated parameters included the number of follicles aspirated, number
and morphology of recovered cumulus-oocyte complexes (COCs), and developmental capacity
of oocytes after in vitro maturation (IVM) and fertilization (IVF). In addition, blood samples
were taken during all OPU sessions and were analyzed for progesterone concentration. To
evaluate the effects of long-term OPU on the subsequent fertility of donors, all heifers were
inseminated during the first natural estrus after the OPU experiment and pregnancy was
determined by ultrasound examination on Day 28 and by palpation per rectum on Day 42 after
insemination.

MATERIALS AND METHODS

Ultrasound-Guided OPU (U-OPU)

The U-OPU technique was essentially conducted as described by Pieterse et al. (17,18)
with an Aloka 500 ultrasound monitor and a UST-974-5 5 MHz sector scanning transducer
(Aloka, Tokyo, Japan). The transducer was extended to a length of approximately 60 cm by a
specifically manufactured grip (Scanning Animal Systems, Stuttgart, Germany) to facilitate
intravaginal manipulation of the transducer tip. A guidance canal (length: 48 em) in the grip of
the transducer made it possible to slide the puncture needle through the transducer.
Theriogenology 91

Laparoscopic OPU (L-OPU)

The L-OPU technique was performed according to Reichenbach et al. (19,20,21) with
minor modifications. The equipment (Dr. Fritz Endoskopie- und Dokumentationssysteme,
Tuttlingen, Germany) consisted of 1) a forward viewing 0° angle telescope (length: 64 cm;
diameter: 11 mm) with an internal canal (diameter: 6 mm, with magnetic valve) to guide the
aspiration needle, attached to an external halogen light source and to a video camera, to control
the puncture of follicles on a television monitor; 2) a 'Cavi-safe' trocar (length: 60 cm; diameter:
11 mm) with safety thorn to prevent injuries to the abdominal organs; and 3) a stainless steel
trocar-sheath (internal diameter: 12 mm; length: 50 cm) with magnetic valve to accommodate
the telescope and a three-way tap at the proximal end of the sheath to enable gas insuffiation into
the abdominal cavity during laparoscopy.

Follicle Aspiration Unit

The aspiration line was the same for both methods and consisted of a single lumen needle
(diameter: 17 G; length: 70 cm; William Cook Europe GmbH, M6nchengladbach, Germany)
connected to a 50-mL Falcon tube by 100-cm Teflon tubing. Vacuum pressure was provided by
a regulated vacuum pump (V-MAR-5000; Cook) and adjusted to create a flow rate of 16 to 20
mL per min. The collection medium consisted of phosphate-buffered saline (PBS) supplemented
with 50 mg/L gentamicin (Sigma, St. Louis, MO), 60 mg/L heparin (Sigma) and 1% fetal calf
serum (FCS; Biochrom, Berlin, Germany).

Experimental Design

To compare the efficiencies of both methods, an experiment was set up in which

Simmental heifers (2 to 2.5 yr old; n = 4 each for treatment Groups A and B; n = 3 each for
treatment Groups C and D, without hormonal stimulation) were subjected to either U-OPU
(Groups A and C) or L-OPU (Groups B and D) twice weekly for 8 wk (15 sessions) followed by
a treatment-Dee interval of 11 wk and another 8-wk period of follicle aspiration using the
respective alternative method. The experiments were done from September 1996 to March 1997
(Groups A and B) and from November 1996 to May 1997 (Groups C and D).
After the OPU experiment, all heifers were inseminated twice with an interval of 12 h in
the first natural estrus and were examined for pregnancy by ultrasound on Day 28 and palpation
per rectum on Day 42 after insemination.

Classification of Recovered Cumulus-Oocyte Complexes (COCs)

The recovered COCs were classified as Class I: 4 or more layers of compact cumulus;
Class II: 1 to 3 layers of compact cumulus; Class III: partially denuded oocytes; and Class IV:
denuded oocytes and/or oocytes showing abnormal cytoplasm.
92 Theriogenology

Evaluation of Developmental Capacity of Oocytes

In vitro maturation (IVM) and fertilization (IVF) of recovered oocytes were carried out as
described previously (23). Briefly, COCs were maintained in tissue culture medium 199 (TCM-
199 with Earle's salts; Seromed, Berlin, Germany) supplemented with 10% heat-inactivated
estrous cow serum (ECS) and 10 lag/mL follicle stimulating hormone (FSH-p; Schering,
Kenilworth, NJ) for 24 h in a humidified atmosphere of 5% COs in air at 39°C.
After IVM, the COCs were transferred to a Tyrode's-Albumin-Lactate-Pyruvate (TALP)
medium containing 6 mg/mL bovine serum albumin (BSA V; Sigma), 10 ~tg/mL heparin
(Sigma) and frozen/thawed semen (10s spermatozoa/mL) that had been subjected to a swim-up
procedure (16). The COCs were maintained in this medium for 18 h under the same conditions
that had been used for IVM.
Cumulus cells were removed from presumptive zygotes by gentle pipetting, and the
embryos were washed 3 times in TCM-199 supplemented with 10% ECS and cultured in this
medium in the presence of cumulus cells at 39°C in a humidified atmosphere of 5% CO2, 5% O2
and 90% N2. The numbers of cleaved embryos and of morulae and blastocysts were recorded at
96 and 168 h after IVF, respectively.

Blood Collection and Measurement of Serum Progesterone Levels of Donors

Blood samples were taken from all donors after each OPU session. Serum was obtained by
centrifugation (2500 x g; 10 min) and stored at -20°C until analysis. Progesterone concentrations
were determined by ELISA as previously described (13,25). Progesterone secretion during the
ftrst and second aspiration period was estimated as area under the curve.

Statistical Analysis

Data were analyzed by Chi-square analysis (overall efficiency of the methods); by Mann-
Whitney U-test (overall efficiency of the methods, variability among donors, progesterone
measurements); and by analysis of variance (GLM procedure; SAS version 6.03; SAS Institute
Inc., Cary, NC). The mathematical model included method (U-OPU vs L-OPU); period (first vs
second OPU-period); the interaction method by OPU-period; and session number (1 to 15,
within period) as covariate. Least squares means (LSMs) and standard errors (SEs) of LSMs
were calculated and compared using Student's t-test.

RESULTS

Overall Efficiency of U-OPU vs L-OPU

The overall numbers of follicles aspirated, oocytes recovered, and embryos produced in
the course of the experiment are summarized in Table 1. The U-OPU procedure could be
successfully performed in all donors throughout the entire experiment (14 donors x 15 sessions
= 210 sessions). The L-OPU method was sometimes difficult or impossible to perform, mainly
due to problems in perforating the fomix vaginae or to intestinal spasms inhibiting correct
Theriogenology 93

positioning of the ovaries. Therefore, only 177 sessions of L-OPU were available for analysis.
All 15 or at least 14 L-OPU sessions were successful in 6 and 4 donors, respectively. The
number of successful L-OPU sessions for the remaining 4 donors corresponded to 4, 8, 9, and
10.

Table 1. Overall efficiency ofU-OPU vs L-OPU

U-OPU L-OPU

Sessions n 210 177

Follicles aspiratedf mean (SD) 5.7 (3.6) a 4.3 (2.4) b
median 5 4
Ooeytes recovered f mean (SD) 3.3 (2.7) ¢ 2.7 (2.1) d
median 3 2
Recovery rate % 58 63
Classification of COCs e

Class I n (%) 269 (38.7) a 100 (21.0) b

Class II n (%) 109 (15.7) 92 (19.3)

Class III n (%) 50 (7.2) a 84 (17.6) b

Class IV n (%) 267 (38.4) 200 (42.0)

Embryos cleavede'g n (%) 404 (58.1)c 248 (52.1)d

Morulae and blastocystse'h n (%) 188 (27.1)a 66 (13.9) b

Embryos per sessionf mean (SD) 0.9 (1.4) a 0.4 (0.6) b

a'dwithin rows proportions marked by different superscripts are significantly different (a:b:
< e • f~ • gh
P<0.001; c:d: P 0.05); Chl-square test, Mann-Whltney U-test. ' Observed at 96 and 168 h
after IVF, respectively.
U-OPU: Ultrasound-guided ovum pick-up; L-OPU: Laparoscopic ovum pick-up.

Variability among Donors

The results shown in Table 2 indicate clear differences between the animals. Comparison
of the 20PU-methods within animal in Groups A and C (donors with U-OPU in the first and L-
OPU in the second period of the experiment) revealed generally greater numbers of aspirated
follicles, recovered oocytes, and developed embryos when using U-OPU than L-OPU. There
was only a single donor with a greater number of oocytes recovered by L-OPU than by U-OPU.
In donors aspirated with L-OPU in the first experimental period and with U-OPU in the
second (Groups B and D), no differences between the 2 methods were found except in a single
94 Theriogenology

donor (Heifer B 09), in which the number of follicles aspirated was significantly greater with L-
OPU than with U-OPU.

Effects of Method, OPU-Period, and Session Number (Analysis of Variance)

Analysis of variance revealed a significant (P<0.05) effect of the aspiration method (U-
OPU vs L-OPU) on the number of Class I (LSMs: U-OPU: 1.28; L-OPU: 0.56) and Class III
oocytes recovered (LSMs: U-OPU: 0.24; L-OPU: 0.45) per session.
The OPU-period had a significant effect on the number of follicles aspirated (LSMs:
Period 1: 5.97; Period 2: 4.00; P<0.01) and on the number of embryos produced (LSMs: Period
1: 0.86; Period 2: 0.42; P<0.05) per session.
A significant effect of the interaction method by OPU-period was found for the number of
follicles aspirated (P<0.001), COC~ recovered (P<0.001), and the number of embryos produced
per session (P<0.01). Furthermore, there were significant (P<0.05) effects of the interaction
method by OPU-period on the proportion of Class I and III COCs.
In the first aspiration period, U-OPU was significantly better than L-OPU with respect to
the number of follicles aspirated, COCs recovered, and embryos produced. Whereas the results
of L-OPU in the second aspiration period were similar to those obtained in Period 1, the
efficiency of U-OPU was reduced in Period 2 and did not differ from that of L-OPU. Both the
LSMs and SEs are shown in Figure 1 (numbers of follicles aspirated, COCs recovered, and
embryos produced) and in Table 3 (proportion of Class I, II, III, and IV COCs).

6.0 ~ a
5.0 . 1.2
b b '~
2.0 0.8

b b
2.0 7= 1.0 0.4
1.0
0.0 0.0 0.0,
Period 1 Period2 Period 1 Pedod2 Pedod 1 Period2

Figure 1. Effect of the interaction method by OPU-period on the number of follicles aspirated
(A), the number of oocytes recovered 03) and the number of embryos produced (C).
The figure shows least squares means (LSMs) and standard errors (SEs) of LSMs.
Values marked by different superscripts are significantly different (Student's t-test;
P<0.01).
U-OPU: Ultrasound-guided ovum pick-up; L-OPU: Laparoscopic ovum pick-up.
Thefiogeno~gy 95

Table 2. Variability among donors

Group/Donor Aspiration Follicles aspirated COCs recovered Embryos produced
Method Mean SD Mean SD Mean SD

A01 U-OPU 10.60 a 4.79 5.87 a 3.74 2.33 a 2.38

L-OPU 2.87b 1.25 1.53b 0.99 0.20 b 0.56

A 02 U-OPU 6.67 a 3.02 2.73 ¢ 1.49 0.73 1.10

L-OPU 1.80 b 1.03 1.20 d 0.79 0.20 0.42
A03 U-OPU 9.93 e 4.03 6.93 e 4.18 1.67 a 1.80
L-OPU 5.20d 2.51 4.27f 2.55 0 .07b 0.26

A04 U-OPU 5.87 e 3.04 3.00 2.07 0.47 0.92

L-OPU 3.27d 1.94 2.47 2.00 0.53 0.83
C05 U-OPU 5.20 2.86 2.53 2.17 0.50 0.85
L-OPU 3.64 2.31 2.50 2.38 0.36 0.84
C 06 U-OPU 6.13 4.61 3.33 e 3.99 1.71 2.37
L-OPU 5.93 1.59 5 -00f 2.04 0.64 0.63
C 07 U-OPU 7.13 e 2.67 4.80 e 2.73 1.79 c 1.42
L-OPU 4.43f 2.14 2-71f 2.13 0.36d 0.63

B08 L-OPU 5.44 2.92 3.00 1.32 0.78 0.67

U-OPU 3.73 1.79 2.53 1.36 0.53 0.64
B 09 L-OPU 5.64 a 2.24 2.29 1.38 0.36 0.63
U-OPU 2.80b 1.42 1.67 1.40 0.27 0.59

B 10 L-OPU 5.00 2.45 3.25 2.22 0.50 1.00

U-OPU 4.60 2.32 3.27 1.75 1.13 1.25
B 11 L-OPU 2.40 1.76 0.93 1.03 0.13 0.35
U-OPU 2.27 0.88 1.40 0.99 0.27 0.59
D 12 L-OPU 4.40 1.88 2.47 : 1.85 0.50 0.65
U-OPU 4.73 1.91 2.13 1.41 0.40 0.51
D 13 L-OPU 5.53 2.42 3.27 2.19 0.50 0.85
U-OPU 5.33 1.59 2.73 1.62 0.40 0.91
D 14 L-OPU 4.88 2.59 3.13 2.42 0.43 0.53
U-OPU 5.00 2.14 3.40 1.50 0.60 0.99

a'fvalues within heifer marked by different superscripts are significantly different (Matin-
Whitney U-test; a:b: P<0.001; c:d: P<0.01; e:f: P<0.05).
U-OPU: Ultrasound-guided ovum pick-up; L-OPU: Laparoscopic ovum pick-up.
96 Thefiogeno~gy

Table 3. Effect of the Interaction Method by OPU-Period on Quality of COCs

Period Aspiration Quality of COCs
Method Class I (%) Class II (%) Class III (%) Class IV (%)
LSM SE LSM SE LSM SE LSM SE
1 U-OPU 32.6a 3.4 17.6 2.8 10.9 a 1.9 38.9 3.8
1 L-OPU 19.4 b 4.1 22.0 3.3 13.1 a 2.3 45.4 4.5
2 U-OPU 47.8 ¢ 3.4 13.9 2.8 3.3 b 1.9 35.0 3.7
2 L-OPU 27.2 ab 3.5 20.0 2.8 15.4 a 2.0 37.4 3.8

a'evalues are means and standard errors. Within column, proportions marked by different
superscripts are significantly different (Student's t-test; P<0.05).
U-OPU: Ultrasound-guided ovum pick-up; L-OPU: Laparoscopic ovum pick-up.

Effects of U-OPU vs L-OPU on Secretion of Progesterone

Total secretion of progesterone during the 2 aspiration periods was estimated as area under
the curve. Progesterone levels were higher during the L-OPU than the U-OPU period; however,
this difference was statistically significant (P<0.05) only for donors first aspirated by L-OPU
(Figure 2).

500

_.--. Groups A and C Groups B and D

400 n°s.

"7
-~ 3oo

200

J_

l
U-OPU L-OPU L-OPU U-OPU

Figure 2. Progesterone secretion (estimated as area under the curve) of donors during the first
and second aspiration period. Box plots are shown with mean (dotted line) and
median values (solid line). The significant (Mann-Whitney U-test; P<0.05) difference
between U-OPU and L-OPU in donors belonging to groups B and D is marked by an
asterisk; n.s. = not significant.
Theriogenology 97

Fertility of Donors After Long-Term OPU

All 14 donors were inseminated twice with an interval of 12 h in the first natural estrus
after the OPU experiment. Seven donors (50%) were pregnant as determined by ultrasonography
on Day 28 and by palpation per rectum on Day 42.

DISCUSSION

The aim of the present study was to investigate whether transvaginal U-OPU or L-OPU is
the more effective method of recovering oocytes for the in vitro production of bovine embryos.
In addition, the advantages and the disadvantages of both methods and the influence of a long-
term OPU-program on subsequent fertility of donors are discussed.
Overall, the mean numbers of follicles aspirated, oocytes recovered and embryos produced
per session were significantly higher with U-OPU than with L-OPU. This may be due to the
different populations of follicles punctured with the 2 methods. The L-OPU technique allows
only for the aspiration of superficial follicles, whereas the U-OPU method also provides for the
recovery of oocytes from follicles deeper in the cortex ovarii. As a consequence more follicles
can be punctured with U-OPU than with L-OPU, and, although there was a nearly identical
recovery rate for both methods in our experiment, a higher number of oocytes was recovered per
session with U-OPU than with L-OPU.
However, there was a significant effect of the interaction method by OPU-period. For
animals of Groups A and C, the OPU-results were significantly better during the first (U-OPU)
than the second (L-OPU) period; whereas for Groups B and D (L-OPU in the first and U-OPU in
the second period), the results were nearly the same in both OPU-periods. This could be due to
the length of the aspiration-free period, which might have been too short to allow for full
recovery of the ovaries. The same reason may account for the fact that the overall number of
follicles aspirated and the number of embryos produced was generally higher in the first than in
the second OPU-period.
There were significant effects of the method and of the interaction method by OPU-period
on quality of COCs recovered by OPU. In agreement with other authors (1) we found, that the
quality of COCs aspirated by U-OPU is better than with L-OPU. Among other factors (i.e.,
different follicle populations obtained by the 20PU-methods), the greater changes in vacuum
pressure during L-OPU vs U-OPU might be responsible for the difference in the quality of
oocytes. This problem may be overcome by a more flexible system for regulating the vacuum
pressure (14).
Following IVM/IVF, the difference in quality of recovered COCs was reflected by the rate
of development to morulae and blastocysts. It was shown that COCs with more than 7 layers of
cumulus cells have a higher developmental competence than COCs with a less intact cumulus
(15). Furthermore, fewer COCs were obtained by L-OPU than by U-OPU, reducing the positive
effects of culturing embryos in groups on their development to morulae and blastocysts (8).
In our experiment, we found a great variability among donors, which was also seen in
other OPU-studies (1,2,3,9,10). However, this variability did not affect our comparison of U-
OPU vs L-OPU since both methods were used for every donor.
98 Theriogenology

During the duration of the experiment blood samples were taken to measure progesterone
concentrations in order to better control the estrus cycle and to determine whether progesterone
levels are influenced by OPU-method. The reason for the higher progesterone levels obtained
during the L-OPU than the U-OPU period may be related to different amounts of bleeding into
the aspirated follicle. The function of straturn granulosum and theca intema is generally not
disturbed by the aspiration of follicular fluid (26). The ability of the theca cells to luteinize and
to secrete progesterone persists as if the follicle had ovulated. But luteinization may be reduced
or be inhibited in hemorrhagic follicles. The deeper penetration by U-OPU affects the ovarian
stroma, thus risking injury to blood vessels. This was confirmed by the presence of more
luteinized follicles during L-OPU than U-OPU, whereas more hemorrhagic follicles were seen
during U-OPU. The observation of additional C.I. during the L-OPU procedure was also made
by other authors (27, 28). Stubbings et al. (24) reported high progesterone levels in the U-OPU
procedure with FSH-treated cows; they attributed these findings to the possible luteinization of
the larger follicles in particular of the stimulated animals.
A pregnancy rate of 50% after a double insemination in the first natural estrus of the
donors following the long-term OPU-program confirms the observation of other authors
(2,4,6,11,20,21) that it is possible to perform OPU at least twice a wk for several wk without
impairing subsequent fertility and state of health of the animals.
In conclusion, both methods of OPU described here were efficacious for the successful
recovery of oocytes from living animals and for in vitro production of bovine embryos.
However, the U-OPU procedure is the less traumatic to the vagina and especially the fomix than
L-OPU, which is confirmed by the fact that U-OPU could be performed successfully in all
donors throughout the entire experiment, whereas L-OPU was sometimes difficult or impossible
to perform. Further, for L-OPU, practical experience is required in order to avoid detachment of
the parietal peritoneum from the vaginal wall or damage the abdominal organs. The risk of
peritoneal infection is also higher for L-OPU than U-OPU. On the other hand, L-OPU allows for
exact positioning of the aspiration needle into the follicle with less trauma to the ovaries. In
addition, with L-OPU there is more information available about vascularization, luteinization
and the content of the punctured follicle prior to aspiration. The overall efficiency of OPU in our
study was relatively low compared with that in other studies (5,19,20,21). This may be due
primarily to our use of heifers. However, using an experimental design that largely excludes the
effects of individual donors and seasonal influences, we can conclude that U-OPU is the more
effective method for the recovery of immature COCs of good quality, thus resulting in a higher
number of embryos following IVM, IVF and IVC.

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