Keywords
in vitro follicular maturation - fertility preservation - vitrification
Palavras-chave
maturação folicular in vitro - preservação da fertilidade - vitrificação
Introduction
Taking into account the recent advances in cancer diagnosis and treatment, young women
diagnosed with cancer present a better prognosis nowadays than a few years ago, with
a good chance of cure or prolonged survival. A large portion of these women still
desire to gestate. Nevertheless, the chemotherapy, the radiotherapy or even the surgery
performed in the treatment may compromise their future fertility.[1]
[2]
[3]
[4]
Cryopreservation of oocytes and embryos are alternatives for young adult women who
can wait ∼ 2–3 weeks for ovarian stimulation and oocyte collection.
The only option for the preservation of the fertility of the patients that require
immediate treatment, such as children and prepubescent adolescents, is the cryopreservation
of ovarian tissue.[1]
[2]
[3]
[4]
[5]
[6]
Studies have shown that freezing ovarian tissue fragments may preserve the gametes
and also the ability to produce hormones.[2]
[7]
[8] The collection procedure can be readily programmed at any phase of the cycle, and
the patient's exposure to hormone treatment is not required.[9]
When the patient is allowed to conceive, thawed fragments could be transplanted again
so that the pregnancy may occur spontaneously or after performing assisted reproduction
(AR) techniques. Studies show that retransplantation is a promising technique, since
60 births have been reported to date.[10]
[11]
Apart from the non-standardization of ovarian tissue cryopreservation techniques,
there are problems such as the local ischemia that occurs after retransplantation,[7] which currently limit the results.
One of the risks of the retransplantation would be the reintroduction of malignant
cells that might have remained in the cryopreserved ovarian tissue.[1] Although to date there are no reports in humans, this is considered a theoretical
risk.[4]
The maturation of existing ovarian follicles in cryopreserved ovarian tissue fragments
in the laboratory to obtain mature oocytes to be fertilized in vitro would avoid the
risk of cancer recurrence.[3]
[4]
[5]
In vitro follicular maturation techniques have been developed for humans, but their
improvement becomes difficult due to the enormous difficulty in obtaining tissue and
due to ethical issues.[12]
The results of human studies are still limited to the study by Amorim et al,[13] in which pre-antral ovarian follicles survived for a period of 7 days after cryopreservation.
Rodrigues et al[14] reported the first in vitro culture of fresh human pre-antral follicles using the
three-dimensional system (3D) in Brazil. The authors obtained follicle growth for
a period of 28 days. Regarding large animals, antrum formation has already been obtained
in cows,[15]
[16] and embryos were produced in sheep[17] and goats.[18] In non-human primates, isolated secondary follicles reached antral stage and were
able to produce mature oocytes for fertilization and embryos.[19] Rodrigues et al[20] obtained embryos of primates in the morula stage from mature oocytes of secondary
ovarian follicles cultured in vitro.
The regulation of the development of the pre-antral follicle is highly complex, and
involves many intra-ovarian and endocrine factors. In this context, more studies are
needed to better understand the mechanisms that control the initiation and development
of follicular growth.[20]
Bovines are widely used as models for the development of these techniques,[12] since their ovaries have large follicle stocks. Furthermore, access to slaughterhouses
is easy, with no ethical restrictions. Similarities in ovarian physiology and dynamics
of the reproductive cycle between humans and bovines make this an excellent experimental
model for reproductive studies.
The main objective of this study was to compare the development of follicles after
vitrification and fresh culture, evaluating the impact of the vitrification in this
process, to contribute to the improvement of the technique and to its future application
in human ovarian follicles.
Methods
We performed a prospective, controlled study approved by the Ethics and Animal Use
Committee of one of our institutions.
Ovary Collection
Bovine ovaries (n = 5) were obtained at a local slaughterhouse in the city of Belo Horizonte, in the
state of Minas Gerais, Brazil. Immediately after the animals were slaughtered, the
ovaries were collected by a properly instructed person and sent to the laboratory
of another of our institutions. The ovaries were placed into a vessel containing glucose
serum and gentamicin (10 μm/mL, Sigma-Aldrich G1264, Sigma-Aldrich, St. Louis, MO,
US) at 4°C. The ovaries were cleaned with water for injection and transferred into
a Petri dish containing α Minimum Essential Medium (αMEM,Sigma-Aldrich M4526) supplemented
with Serum Substitute Supplement (SSS, Irvine 99193, Irvine Scientific, Santa Ana,
CA, US), and gentamycin (10μm/mL).
Ovariy Processing, Follicle Isolation, Encapsulation and Culture
Each ovary was cut in half with the aid of a Solidor scalpel blade #24 (Lamedid, Osasco,
SP, Brazil). One half of each ovary was placed into a tissue slicer, in which it was
possible to remove a thin layer of the ovarian cortex. The medulla was discarded.
Using scalpels, curve scissors and tweezers, we obtained 24 fragments of 2 × 3 × 0.5 mm
from the ovarian cortex.
Total 12 of these fragments were separated and kept in a holding medium (containing
αMEM supplemented with SSS, and gentamycin 10 μm/mL) inside an incubator at 37°C and
5% carbon dioxide (CO2) until the time of vitrification. The remaining twelve fragments were subjected to
the enzymatic and mechanical follicle isolation procedures.
These twelve fragments were separated on four conical tubes (BD Falcon 352099, Becton,
Dickinson and Company [BD], Franklin Lakes, NJ, US) of 15 mL (3 fragments per tube)
containing the holding medium and collagenase (1 mg/mL type IA, Sigma-Aldrich C5894).
The tubes were placed into a water bath for 20 minutes. Every 5 minutes the tubes
were removed from the bath and vortexed for 3 minutes. After 20 minutes, the tubes
were centrifuged for 5 minutes at 1,500 rpm. After removing the supernatant, 3 mL
of a manipulation medium at 4°C was added to stop digestion. The centrifugation process
was repeated once again, the supernatant was removed, and 1 mL of the medium was added.
The fragments were then kept incubated at 37°C, with 5% CO2 and pH between 7.2–7.4. The strips were removed individually and placed into a new
plate with the holding medium to perform mechanic follicle isolation under a stereomicroscope
magnifying glass.
With the aid of 25 g needles, secondary follicles with diameters between 125 μm and
400 μm were isolated from each fragment. These follicles were transferred into another
dish with the holding medium, and placed in the incubator at 37°C for a period of
2 to 6 hours, during the processing of all ovarian strips. The isolation of a total
of 51 fresh secondary follicles was possible.
Only secondary follicles with the following characteristics were selected for encapsulation:
absence of antral cavity; an intact basement membrane, with attached stroma; a visible
rounded and centrally-located oocyte within the follicle.
Drops of 10 µL of sodium alginate 0.25% (FMC Biopolymers, Philadelphia, PA, US) were
prepared. Each follicle was washed individually in two of these drops, and kept into
the third and final drop. With a 10-μL tip, the alginate drop containing the follicle
was pipetted and gently placed on a cross-linking calcium solution. The process was
repeated for all of the isolated follicles.
The cross-linking solution (50 mM CaCl2, 140 mM NaCl, and 10 mM HEPES solution) is responsible for the chemical reaction
that allows the liquid sodium alginate 0.25% to be transformed into gel.
After one minute in the solution, the alginate gel structures where the follicles
were located were transferred onto individual wells of a 48-well culture dish (BD
Falcon 353872) containing 300 μL of αMEM supplemented with 1,050 ng/mL of follicle
stimulating hormone (FSH, Sigma-Aldrich F2293), 3 mg/mL of albumin (Irvine Scientific
9988), 100 mg/mL of fetuin (Sigma-Aldrich F6131), 1 mg/mL of insulin (Sigma-Aldrich),
1 mg/mL of transferrin (Sigma-Aldrich T8158), and 50 ng/mL sodium selenite (Sigma-Aldrich
S5261).
The encapsulated follicles were cultured for a period of 20 days.Every 2 days, 150 uL
of the culture medium of each well of the plate was replaced with fresh medium. During
the culture, images of each of the follicles were obtained to measure their diameter
using the ImageJ software 1.33U (National Institutes of Health, Bethesda, MD, US).
Vitrification and Warming
The fragments were vitrified using the method described by Ting et al (2011).[21] Initially, the fragments were equilibrated sequentially in solutions containing
1.2 M of glycerol (10% glycerol v/v, Sigma-Aldrich G2025) for 3 minutes, 1.2 M of
glycerol + 3.6 M of ethylene glycol (10% glycerol + 20% ethylene glycol Sigma-Aldrich
102466) for 3 minutes and 3 M of glycerol + 4.5 M of ethylene glycol (25% glycerol + 25%
ethylene glycol) for 1 minute. This whole procedure was performed at room temperature.
Following the last solution, the fragments were placed individually onto a piece of
aluminum foil (measuring 8 × 4 mm2) and immediately submerged into liquid nitrogen (−196°C), transferred into cryovials,
and stored until the moment of thawing. For warming, the cryovials were removed from
the liquid nitrogen, and the fragments were individually placed immediately for 5
minutes into solutions containing 0.5 M of sucrose (Sigma-Aldrich S1888), then 0.25 M
of sucrose, 0.125 M of sucrose and, to finish, only on equilibrium solution or holding
medium by 2 times of 10 minutes, and the whole procedure was performed at a temperature
of 37°C. After warming, the fragments were kept in the holding medium solution supplemented
with 15% v/v SSS and 29 mg/mL of ascorbic acid (Sigma-Aldrich A4403) to isolate its
secondary follicles and individually encapsulated into the alginate.
After warming, the fragments were processed using the same protocol performed for
the fresh ones: collagenase enzyme isolation, mechanical procedure, encapsulation
in alginate gel, and culture for 20 days.
Follicle Survival and Growth
The survival and diameters of the follicles were evaluated using images taken with
an Eclipse Ti-S inverted microscope (Nikon, Tokyo, Japan) and an attached digital
camera (OCTAX Microscience, Bruckberg, Bavaria, Germany). Images were obtained from
each follicle, as well as images from a micrometer (1 mm with 0.01 mm divisions, Fisher
Micromaster, Fisher Scientific, Fair Lawn, NJ, US) for calibration purposes. For the
measurement, the images were imported to the ImageJ software 1.33U. Each follicle
diameter was determined in units of pixels and converted to micrometers based on the
conversion determined by measuring the image of the calibrated micrometer.
The follicles were measured from the outer layer of the cells, and the measurements
included the largest follicle diameter and a second measurement perpendicular to the
first. The mean of these values was then calculated and considered as the follicle
diameter.Antrum formation was observed upon a morphological analysis of the follicles.
The follicles were considered to be degenerating when the oocyte was no longer surrounded
by a layer of granulosa cells, and when the oocyte became dark and the granulosa cells
became dark and fragmented.
Statistical Analysis
The statistical analysis consisted of absolute and relative frequencies for categorical
variables and mean ± standard deviation (SD) for continuous variables. The comparison
of two proportions was performed by proportions tests; the comparison of the percentage
of antrum formation, by Fisher exact test; and the comparison of two means, by nonparametric
Wilcoxon test.
In order to evaluate the growth of the ovarian follicles, we used the percentage variation
between a start time T0
and an end time T1
, calculated by the formula: V = (T1-T0)/T0 x 100.
The Kaplan-Meier method and log-rank test were used to estimate the curves until the
infeasibility of the follicles, and to compare them according to their groups (fresh
and vitrified), and we calculated the median survival time of the follicles considering
the follicle survival time, and the number of days between the isolation and their
infeasibility. The analyses were performed using the free R software (R Foundation
for Statistical Computing, Vienna, Austria), version 3.1.3. The significance level
was set to 5%.
The power of the sample was of 95%, and the calculation was based on the size of the
sample analyzed and the proposed objective.
Results
In the present study, 61 follicles were recovered after treatment with collagenase.
A total of 51 follicles were isolated from fresh tissue, and 10 from vitrified tissue.
The survival rate at the end of 20 days of culture was 43.1% for the fresh group,
and 20% for the vitrified group, without any significant difference.
[Table 1] shows the distribution of the surviving follicles on days 1, 5, 10, 15 and 20, regarding
the fresh and vitrified groups, and the comparison test between the proportions of
survivors per day. In absolute terms, the proportions of fresh surviving follicles
were higher than those of the follicles after vitrification in all evaluated days,
but without any significant difference. We were able to observe a gradual reduction
in survival in the two groups, mainly in the vitrified group, though it was not significant.
Table 1
Distribution of surviving follicles per day and comparison between the groups
|
Day
|
Group
|
N
|
%
|
p
|
|
1
|
Fresh
|
51
|
100
|
–
|
|
Vitrified
|
10
|
100
|
|
5
|
Fresh
|
49
|
96.1
|
0.421
|
|
Vitrified
|
9
|
90.0
|
|
10
|
Fresh
|
31
|
60.8
|
1.000
|
|
Vitrified
|
6
|
60.0
|
|
15
|
Fresh
|
24
|
47.1
|
0.741
|
|
Vitrified
|
4
|
40.0
|
|
20
|
Fresh
|
22
|
43.1
|
0.290
|
|
Vitrified
|
2
|
20.0
|
Note: p value refers to the Fisher exact test.
[Fig. 1] shows Kaplan-Meier curves for follicle survival time regarding both groups. We observed
that the fresh follicles survived longer; however, the difference was not significant
(p = 0.205).
Fig. 1 Kaplan-Meier curves and log-rank test for the survival time of the fresh and vitrified
follicles in days; (A) only the curves and (B) curves with confidence intervals.
[Table 2] presents the mean ± SD of the diameters (in µm) of the two groups of follicles per
day. Considering the follicles that survived, Table 2 also shows the mean diameters
of both groups on the first and last days of culture. We observed a significant difference
between the two groups on the first day of culture.
Table 2
Mean fresh and vitrified follicle diameters (in µm) according to the days following
isolation
|
Day
|
Fresh
|
Vitrified
|
p
|
|
Mean ± SD
|
Mean ± SD
|
|
1
|
244.55 ± 80.57
|
348.78 ± 88.73
|
0.002*
|
|
5
|
327.94 ± 87.84
|
375.35 ± 115.83
|
0.209
|
|
10
|
385.43 ± 77.98
|
426.59 ± 104.29
|
0.385
|
|
15
|
411.96 ± 87.77
|
485.70 ± 132.73
|
0.262
|
|
20
|
412.99 ± 102.55
|
422.93 ± 85.05
|
0.725
|
Abbreviation: SD, standard deviation.
Notes: p value refers to the Wilcoxon test. *Represents statistical difference (p < 0.05).
During culture, the vitrified group showed a variation in growth of 8.8% compared
with 21.5% for the fresh group ([Table 3]).
Table 3
Mean variation (in %) of the fresh and vitrified follicle diameters
|
Days
|
Fresh
|
Vitrified
|
p
|
|
Mean ± SD
|
Mean ± SD
|
|
1–5
|
41.7 ± 39.6
|
7.6 ± 7.5
|
0.000*
|
|
5–10
|
8.9 ± 12.7
|
13.7 ± 8.4
|
0.186
|
|
10–15
|
3.2 ± 9.4
|
9.9 ± 4.0
|
0.042*
|
|
15–20
|
1.3 ± 13.0
|
5.9 ± 8.3
|
0.587
|
|
Total
|
21.5 ± 21.4
|
8.8 ± 6.3
|
0.074
|
Abbreviation: SD, standard deviation.
Notes: p value refers to the Wilcoxon test. *Represents statistical difference (p < 0.05).
We observed a significant difference in variation of follicle growth between the groups
from the first to the fifth days (p = 0.000). The fresh follicles showed a higher growth during this period. After vitrification,
the follicles showed a significantly higher growth during the period from the tenth
to the fifteenth days (p = 0.042). In general, the difference in growth between the two groups was not significant.
[Fig. 2] shows the distribution of antrum formation in both groups. We observed that the
fresh follicles showed a higher mean antrum formation (47.1%) when compared with the
vitrified follicles (20%), but this difference was not significant (p = 0.167).
Fig. 2 Distribution of antrum formation for the fresh and vitrified groups. Note: p = 0.167.
Discussion
The results obtained demonstrated that the follicles cultured in vitro after vitrification
are able to survive, develop and form antrum.
These are the first data (to the best of our knowledge) of isolation and follicle
maturation obtained from vitrified bovine ovarian tissue.
Despite the sample size, we obtained a survival rate and antrum formation of 20% of
these follicles following 20 days of in vitro culture in an alginate matrix, which,
for an initial study, is a great advance. However, we must highlight the need for
further studies in order to reach a consistent conclusion.
The viability of both follicle groups, overall, showed no significant differences.
As described by Bulgarelli et al,[22] macaque secondary follicles isolated from vitrified ovarian tissue showed similar
survival rates when compared with the fresh group, and showed no significant difference
in the diameter and antrum formation after in vitro culture in the 3D system.
In a study by Amorim et al[13] that evaluated the survival of human pre-antral follicles after ovarian tissue cryopreservation,
follicle isolation and in vitro culture into an alginate matrix, the authors did not
observe any morphological or viability changes among the groups: follicles included
in fresh ovarian tissue fragment, follicles included in the cryopreserved tissue,
enzymatically-isolated follicle fragments after cryopreservation, and fresh follicles
cultured in an alginate matrix. This study proved to be the first in which the in
vitro culture of human small pre-antral follicles in cryopreserved ovarian tissue
after isolation and culture for 7 days was possible.
In our study, a progressive growth in follicle diameter during the 20 days of culture
was observed. This suggests that the use of a 3D culture system effectively simulates
the physiological conditions, promoting interactions between somatic cells, which
may be an adequate support for the in vitro maturation of isolated pre-antral follicles.[4]
[23]
Xu et al[24] showed that, from fresh human ovarian tissue, they obtained the survival and growth
of isolated secondary follicles using a 3D matrix. Therefore, they were able to create
an environment that enabled oocyte growth.
In a study by Silva et al,[25] higher survival rates in goat secondary follicle cultures using the 3D system were
obtained when compared with the two-dimensional (2D) system.
Another important factor to support follicular development was the use of a culture
medium with an adequate composition to aid the development.
In the present study, a culture medium containing FSH was used for both groups. The
FSH is considered important for the in vitro maturation of follicles. Its absence
may contribute to low antrum formation rates, as demonstrated by Luz et al[26] and Barberino et al.[27] In a study published by Matos et al,[28] a concentration of 50 ng/mL of FSH was able to promote the growth of activated pre-antral
follicles, demonstrating that the FSH plays a vital role in the culture of goat ovarian
follicles.
However, to date, there are no conclusive studies on the effectiveness of different
media for the in vitro culture of bovine ovarian tissue, especially after cryopreservation.[29]
In the present study, we also found that the diameters of the vitrified follicles
on the first day of culture were significantly higher than those of the fresh follicles
(p = 0.002). We believe that this fact may be related to the use of collagenase. This
enzyme in fresh ovarian tissue assists in the isolation of pre-antral follicles, leaving
the tissue less dense, and the follicles more accessible within the stroma, which
makes the removal of the stromal cells surrounding the teak easier.
On the other hand, the vitrified follicles showed a higher fragility, which hampered
their removal, leaving a thicker layer of stromal cells on their surfaces, making
it difficult to measure their diameters on the first day of culture. This difficulty
in isolating the follicles after vitrification also explains the lower number of follicles
collected in this group.
During our culture, the fresh follicles showed a higher growth from days 1 to 5 when
compared with the vitrified follicles during the same period. On the other hand, the
vitrified follicles grew more between days 10 to 15 of culture. We obtained a variation
in diameter growth of 21.5% for the fresh group versus 8.8% for the vitrified group
(p = 0.074), and a mean diameter of 412.99 μm and 422.93 μm respectively (p = 0.725).
The reason for this variation in growth is unknown; however, Gutierrez et al,[15] in an in vitro culture of fresh bovine pre-antral ovarian follicles, indicated a
rapid increase in follicle diameter during the first week of culture, followed by
a delay in growth after 8–10 days of culture. This finding is in agreement with the
results found in the present study.
We can also assume that the higher growth of vitrified follicles within 10 to 15 days
could be justified since these follicles were the most resistant follicles selected
in the vitrification and isolation processes: they were able to maintain their structural
integrity during vitrification and isolation, and had higher survival capacity.
Moniruzzaman et al[30] observed a slowdown in the rate of development of vitrified primordial follicles
of pigs. Ting et al[5] also observed a delay in the growth of vitrified isolated secondary follicles of
primates.
Another possibility would be the alleviation of the effect of stromal cells in the
measurement of follicular diameter during culture. Thus, the growth rate could have
been similar, but confused by the larger initial diameter.
After warming, the tissue was exposed to digestion by collagenase, followed by mechanical
isolation as performed in the fresh method. After it was digested, the vitrified tissue
showed greater sensitivity to the mechanical isolation, resulting in easy follicle
rupture. These findings can be related to the enzymatic method of isolation of ovarian
follicles in vitrified tissue.
The enzymatic isolation is used to facilitate the isolation of follicles with dense
and fibrous ovarian cortices, such as those of humans.[31] The enzymatic isolation of pre-antral follicles improves the recovery rate of the
isolated follicles; however, the integrity of the follicle is not always preserved,
damaging the theca cells and the basal membrane.[31]
[32]
[33]
Rupture in the follicular membrane and the destruction of other intrafollicular components
during the enzymatic isolation represent major problems in the development of pre-antral
follicles in vitro.[34] The potential follicle development may be hindered if the integrity of the follicle
is compromised.[4]
The sensitivity of the ovarian cortex fragments demonstrated that the use of collagenase
after vitrification could damage intact follicles for in vitro culture in the 3D matrix
systems, but the small sample size is a limitation. Moreover, vitrification has a
direct effect on the quality of the tissue after the process.
Vitrification is a challenging process due to the complex structure and different
cell types present in the ovarian tissue,[35] even though it is considered an effective, faster and less expensive process.
Ting et al[21] demonstrated that macaque secondary follicles are better preserved after vitrification
when compared with slow freezing.
In a recent study, Lunardi et al,[36] comparing the effect of vitrification in the development of sheep secondary follicles
included in the ovarian tissue versus follicles isolated using only mechanical isolation, verified that follicles that
were vitrified included in the tissue showed significantly larger rate of follicles
that regressed its growth.
According Campos et al,[8] the cooling and warming processes may cause irreversible damage to the ovarian tissue.
Another important factor that is related to the number of isolated follicles is that
each tissue fragment presents a different number of follicles, which makes the comparison,
in terms of numbers of follicles, of the vitrified and fresh tissue groups very difficult.
Even though the effects of collagenase and/or vitrification may impair the isolation
of pre-antral follicles, the growth and development in an in vitro culture was possible,
confirming the viability of the vitrified follicles after enzymatic isolation.
The follicles that have survived until the last day of culture, in 100% of the cases,
reached the antral stage. Antrum formation was observed from the 4th day of culture
in most follicles. This ability to develop and form antrum can be considered a follicular
functionality indicator.[16]
In the present study, the ovarian tissue was subjected to several different procedures,
such as vitrification, warming, follicular isolation and encapsulation in alginate
gel. These procedures may potentially damage the follicles, as discussed by Amorim
et al[37] and Dolmans et al.[38]
Based on our results, we can assume that the isolation and culture of bovine ovarian
follicles after vitrification is a possible approach. We believe more studies are
necessary to improve the in vitro culture of follicles, especially for tissues previously
submitted to cryopreservation. These preliminary results are important to increase
the understanding and the chances of future applicability in patients requiring immediate
treatment for cancer, as well as in children and prepubescent adolescents.
Conclusions
In conclusion, the present study demonstrates, for the first time, the feasibility
of in vitro follicular maturation of bovine secondary pre-antral follicles after vitrification
and enzymatic and mechanical isolation from ovarian tissue fragments in a 20-day period
in an alginate matrix. Despite the lower results obtained with the fresh tissue, the
cultured follicles of vitrified tissue were able to grow and, at the end of the culture,
100% of the survivors formed antrum.
The enzymatic isolation with collagenase may have impaired the efficiency in the isolation
of a higher number of follicles, which indicates the need for further studies to evaluate
the best technique to be used.
Despite the difficulty with the technique, we can conclude that the 3D culture system
is potentially viable, and provides a new opportunity to study the optimization of
in vitro follicle maturation, as well as the future possibility of clinical application.
