Introduction
Endometriosis is a benign gynecological disease characterized by the presence and
growth of endometrial tissue (glands and stroma) outside the uterus.[1 ] This disease is associated with infertility: ∼ 30% of infertile women present endometriosis[2 ] and between 30 and 50% of women with endometriosis have difficulties in becoming
pregnant.[3 ]
In cases with advanced endometriosis (moderate or severe, stage III/IV), infertility
could be due to pelvic anatomical alterations caused by lesions and adherences.[4 ] On the other hand, minor endometriosis (minimal or mild, stage I/II) is not associated
with marked changes in the pelvic anatomy (American Society for Reproductive Medicine
[ASRM], 1997)[4 ] It is unclear whether, after assisted reproduction technologies (ART), endometriosis
has a negative impact on clinical pregnancy and live birth rates.[5 ]
[6 ]
[7 ] Findings from the most recent meta-analysis showed that women with and without endometriosis
have comparable ART outcomes in terms of live births, whereas those with severe endometriosis
have inferior outcomes.[6 ] On the other hand, classical studies assessing natural conception reported lower
cumulative pregnancy rate in women with early-stage endometriosis compared with women
with infertility of unknown cause.[8 ]
[9 ]
In endometriosis women, menstrual reflux and macrophages have been implicated as potential
inducers of oxidative stress (OS)[10 ]
[11 ] which, in turn, is involved in impairment of oocyte quality, and in compromising
the reproductive capacity of women with early-stage endometriosis.[12 ]
[13 ]
Previous studies demonstrated that follicular fluid (FF) from infertile women with
mild endometriosis (ME), when added during in vitro maturation (IVM) causes chromosome
misalignment and meiotic spindle alterations in bovine[14 ]
[15 ] and murine[16 ] oocytes. Alterations in oocytes during IVM may affect in vitro embryo development.[17 ]
[18 ] However, no study to date has evaluated the effect of FF from infertile women with
ME on embryo development and the impact of antioxidants on this response.
L-carnitine (LC) is a lysine derivative that clears hydrogen peroxide and products
of lipid peroxidation.[19 ] In mitochondria, LC also facilitates the transport of fatty acids during β-oxidation,
a major pathway for Adenosine Triphosphate (ATP) production.[20 ] N-acetyl-cysteine (NAC) is an amino thiol with immunomodulatory, anti-apoptotic,
and antioxidant properties.[21 ] N-acetyl-cysteine is a precursor of intracellular cysteine and reduced glutathione
(GSH), which also is and intracellular antioxidant.[22 ]
As human oocytes and embryos are extremely rare, and their use in invasive studies
usually prevents their subsequent use in ART, studies using animal models may be useful
for the elucidation of the mechanism by which endometriosis leads to infertility.
Our hypothesis is that ME leads to OS, and this leads to infertility due to an impairment
of oocyte and embryo quality. Therefore, the aim of the present study was to evaluate
the impact of adding FF from infertile women with ME and without endometriosis (control),
and antioxidants (NAC and/or LC) to the IVM medium of bovine oocytes on in vitro embryo
development (cleavage, blastocyst formation and hatching).
Methods
The present experimental study used an in vitro bovine model. The present study was
approved by the Ethics Committee for Animal Experimentation of the Faculdade de Medicina
de Ribeirão Preto, Universidade de São Paulo (FMRP-USP, in the Portuguese acronym)
(n° 169/2008) and the Research Ethics Committee of the University Hospital, FMRP-USP
(n° 12201/2008).
Patient Selection and Follicular Fluid Collection
Twenty-two FF samples were obtained between February 2009 and February 2011 from infertile
women who underwent ovarian stimulation for intracytoplasmic sperm injection (ICSI)
at the Sector of Human Reproduction, Department of Gynecology and Obstetrics of the
FMRP-USP.
The endometriosis group consisted of patients with infertility associated exclusively
with ME, without other gynecological or clinical conditions. An experienced surgeon
diagnosed and classified these women by videolaparoscopy using the criteria of the
American Society for Reproductive Medicine (1997).[4 ] The control group consisted of women with tubal or male factor infertility. All
control women also underwent videolaparoscopy as part of the protocol for investigation
of marital infertility. None of the controls had endometriosis or any other gynecological
diseases.
The exclusion criteria were established to reduce confounding factors that could affect
OS and/or oocyte quality. Thus, women with any of the following conditions were excluded:
age ≥ 38 years old; body mass index (BMI) ≥ 30 kg/m2 ; serum concentration of follicle stimulating hormone (FSH) on the 3rd day of the menstrual cycle ≥ 10 mIU/mL; chronic anovulation; presence of hydrosalpinx
or chronic diseases such as diabetes mellitus or any other endocrinopathy; cardiovascular
disease; dyslipidemia; systemic lupus erythematosus or any other rheumatologic disease;
HIV infection or any other active infection; smoking; and use of vitamins, hormonal
or nonhormonal medications during the 6 months before inclusion in the study.
Comparison of the means and standard errors of the means (SEMs) indicated that the
endometriosis and control groups had similar age (32.72 ± 0.52 versus 30.63 ± 1.36
years old), FSH concentration on the 3rd day of the menstrual cycle (5.02 ± 0.90 versus 5.79 ± 0.62 mIU/mL), number of follicles
measuring between 14 and 17 mm (10.09 ± 1.43 versus 6.11 ± 1.52 mm), and number of
follicles of at least 18 mm after ovarian stimulation (4.89 ± 0.72 versus 3.11 ± 0.76 mm).
Protocol of Controlled Ovarian Stimulation
Controlled ovarian stimulation (COS) was performed according to our institutional
protocol (long protocol). Pituitary blockade was performed by administering an agonist
of gonadotropin releasing hormone (GnRH) (Lupron, Abbott, São Paulo, SP, Brazil).
Controlled ovarian stimulation was performed by administering recombinant FSH (Gonal-F,
Serono, Geneva, Switzerland; Puregon, Organon, Oss, The Netherlands), and ovulation
was induced with human chorionic gonadotropin (hCG) (Ovidrel, EMD Serono, Rockland,
MA, USA).
Each patient received a daily subcutaneous injection of 0.5 mg leuprolide acetate
(Lupron; Abbott) starting 10 days after the first ultrasound exam before COS. Recombinant
FSH (Gonal-F; Puregon, 200–225 units/day) was administered during ovarian stimulation,
and follicular growth was monitored. Ovulation was triggered with Ovidrel, and oocytes
were retrieved between 34 and 36 hours later.
Collection and Processing of FF Samples
Follicular fluid was collected into individual sterile tubes preheated to 37°C in
the absence of culture medium. The sample was only from the first follicle (mean diameter ≥ 15 mm)
of the first ovary punctured, with aspiration of the full follicular content. Only
FF with no blood contamination upon visual inspection and with a mature oocyte was
used. The samples were centrifuged at 300 g for 10 minutes to remove the remaining
cells, and the supernatant was stored at - 80°C in two aliquots for future use. Follicular
fluid was collected from 22 infertile women, 11 with ME and 11 with male and/or tubal
infertility.
We pooled the 11 FF samples of each group for experiments because we had previously
tested these samples individually in a study of the role of FF from women with infertility
related to ME.[14 ] The results of that study indicated no intragroup differences and a homogeneous
response in all 11 samples from each group. This previous study[14 ] also tested the effect of 4 different FF concentrations added to the IVM medium
(1%, 5%, 10%, and 15%) and indicated no dose-dependent effect. Thus, we used 1% FF
concentration.
Chemicals and Reagents
All chemicals and reagents were purchased from Sigma Chemical Co (St. Louis, MO, USA),
unless otherwise stated.
Preparation of Antioxidant (N-acetyl-cysteine and L-carnitine) Solutions
The solutions of both anti-oxidants were prepared at 100 × (150 mM NAC and 60 mg/mL
LC) using water after passage through a filter with 0.22 μm pore. The NAC concentration
used to supplement the IVM was 1.5 mM,[23 ] and the LC concentration was 0.6 mg/mL.[18 ]
Oocyte Collection
Bovine ovaries were collected immediately after slaughter and transported in physiological
saline maintained at between 35 and 38.5°C. In the laboratory, the ovaries were washed
with physiological saline supplemented with an antibiotic, and follicles measuring
2 to 8 mm were aspirated by a 21-gauge needle mounted on a 10-mL syringe. Cumulus-oocyte
complexes (COCs), with homogeneous cytoplasm and at least 4 layers of cumulus oophorus
cells, corresponding to grades 1 and 2 as described previously,[24 ] were selected under a stereomicroscope and washed in holding medium (TCM-199 medium
containing Hanks salts, HEPES buffer, and L-glutamine (Invitrogen, Gibco Laboratories
Life Technologies, Thermo Fisher Scientific, Waltham, MA, USA).
In Vitro Maturation
Selected COCs were cultured in plates containing 4 wells (NUNC, Thermo Fisher Scientific,
Waltham, MA, USA) in groups of 20 per well, with 400 µL of culture medium for IVM
at 38.5°C, 95% humidity, and 5% CO2
[17 ]
[25 ] in a culture system without mineral oil for between 22 and 24 hours. The IVM medium
was TCM-199 with Earle salts and bicarbonate (Invitrogen, Gibco Laboratories Life
Technologies, Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 0.4 mM
sodium pyruvate, 0.5 µg/mL gentamicin, 5 µg/mL FSH, 5 mg/mL LH, 1 µg/mL estradiol,
and 10% fetal calf serum (FCS) (Gibco Laboratories Life Technologies, Thermo Fisher
Scientific, Waltham, MA, USA)
In Vitro Fertilization
In vitro matured oocytes were fertilized in vitro with frozen semen from a single
bull (CRV Lagoa, SP, Brazil). Before addition, the frozen semen was thawed in 35°C
water for 30 seconds. Swim-up was realized as described by Parrish et al.[26 ]
[27 ]
Briefly, the COCs were gently pipetted to remove adhering granulosa cells and to break
apart aggregations. The disaggregated COCs were transferred into 50 μL microdrops
of fertilization medium (114 mM NaCl, 3.1 mM KCl, 25 mM NaHCO3, 47 mg/L NaH2PO4.H2O,
10 mM HEPES, 10 mM sodium lactate 60%, 1.4 mM caffeine, 0.5 mM MgCl2.6H2O, 2 mM CaCl2.2H2O,
0.4 mM sodium pyruvate, 0.5 µg/mL gentamicin, 6 g/L BSA,10 µg/mL heparin, and 40 µL/mL
each of penicillamine, hypotaurine, and epinephrine). Sperm (1 × 106/mL) was added
and the medium was maintained for between 18 and 22 hours at 38.5°C in a humidified
incubator in air with 5% CO2.
In Vitro Embryo Culture
The presumptive zygotes were denuded from cumulus cells by gentle pipetting and cultured
in 50 μL droplets in CR2aa medium supplemented with 1 mg/mL BSA, 40 μg/mL sodium pyruvate,
5 mg/mL hemi-calcium, 20 μL/mL amino acid solution (0.09 μg/mL glutamine, 0.15 μg/mL
alanine, and 0.75 μg/mL glycine), 20 μL/mL Eagle Basal Medium (EBM), 10 μL/mL Eagle
Minimum Essential Medium (MEM) and 5% FBS, at 38.5°C in a humidified incubator in
air with 5% CO2. The culture was maintained for 9 days after fertilization and the
medium was renewed every 2 days.
The number of cleaved embryos was recorded on day 3 (72 hours after insemination [HAIs]),
blastocyst production was counted on day 7 (168 HAIs), and hatching of blastocysts
was checked on day 9 (216 HAIs).
Experimental Design
Immediately after selection, the COCs were subjected to IVM for between 22 and 24
hours, and divided in groups according to the supplementation of IVM medium with FF
and antioxidants according to [Figure 1 ] ([Fig. 1 ]). After between 22 and 24 hours of IVM, IVF was performed as described above, and
embryos were cultured in vitro to assay cleavage, blastocyst formation, and hatching
rates.
Fig. 1 Experimental design.
Statistical Analysis
Data were analyzed using RStudio software (R Foundation, Vienna, Austria). Categorical
variables (cleavage, blastocyst formation and hatching) were expressed as percentage
and they were compared by the chi-squared test, considering p < 0.05.
Results
We performed IVF on 898 mature COCs and then cultured 883 presumptive zygotes in vitro.
[Table 1 ] shows preimplantation embryo development as cleavage rate, blastocyst formation
rate and hatching rate of the different groups.
Table 1
Embryo development after in vitro fertilization of bovine oocytes which underwent in vitro maturation in medium without follicular fluid (No-FF), with 1% FF from infertile
control women (CFF), or with 1% FF from infertile women with mild endometriosis (MEFF).
The media with CFF and MEFF were supplemented with no antioxidants, 1.5 mM N-acetyl
cysteine (NAC), 0.6 mg/mL L-carnitine (LC), or both antioxidants (NAC+LC)
No − FF
CFF
CFF + NAC
CFF + LC
CFF + NAC + LC
MEFF
MEFF + NAC
MEFF + LC
MEFF + NAC + LC
p -value
Presumptive zygotes (n )
100
96
98
98
101
93
97
100
100
Cleavage rate % (n )
65.00% (65)
61.46% (59)
57.14%
(56)
56.12%
(55)
60.40%
(61)
50.54%
(47)
57.73%
(56)
56.00%
(56)
51.00%
(51)
p = 0.5376
Blastocysts formation rate % (n )
38.00%
(38)
29.17%
(28)
26.53%
(26)
34.69%
(34)
32.67%
(33)
25.81%
(24)
37.11%
(36)
32.00%
(32)
26.00%
(26)
p = 0.4249
Hatching rate % (n )
42.10%
(16)
42.86%
(12)
50.00%
(13)
32.35%
(11)
39.40%
(13)
12.50%
(3)a
44.44%
(16)
18.75%
(6)b
30.77%
(8)
a vs No-FF p = 0.029 vs CFF p = 0.036 vs MEFF + NAC p = 0.020
b vs MEFF + NAC p = 0.045
Note: Data were obtained from 5 replicates. Letter indicate p value < 0.05 (chi-square test).
a
MEFF vs No-FF (p = 0.029), vs CFF (p = 0.036), vs MEFF+NAC (p = 0.02).
b MEFF+LC vs MEFF+NAC (p = 0.045).
No differences were observed between groups in cleavage (p = 0.5376) and blastocysts formation (p = 0.4249) rates ([Table 1 ]).
However, in relation to the hatching rate, the groups without FF (No-FF: 42.10%) and
with FF from control women (CFF: 42.86%) had a higher hatching rate than the group
with FF from ME women (MEFF: 12.50%, versus No-FF: p = 0.029; versus CFF: p = 0.036).
Addition of antioxidants in groups with FF from control women did not alter the hatching
rate [CFF versus CFF + NAC (50.0%, p = 0.800), versus CFF + LC (32.35%, p = 0.557) and versus CFF + NAC + LC (39.40%, p = 0.990)].
Addition of NAC in groups with FF from ME women increased hatching rate (MEFF versus
MEFF + NAC: p = 0.020), being more efficient than the addition of LC (MEFF + NAC versus MEFF + LC:
p = 0.045). However, addition of LC and NAC + LC did not alter the hatching rate in
groups with FF from ME women (MEFF versus MEFF + LC [18.75%, p = 0.793] and versus MEFF + NAC + LC [30.77%, p = 0.224]).
Hatching rates in the group with FF from ME women plus the addition of LC or LC + NAC
were similar to the groups without FF (No-FF versus MEFF + LC: p = 0.066 and versus MEFF + NAC + LC: p = 0.511) and with FF from control women (CFF versus MEFF + LC: p = 0.080 and versus MEFF + NAC + LC: p = 0.524).
Discussion
The etiopathogenesis of ME-related infertility remains unclear. The present study
is the first to show that FF from infertile women with ME impairs bovine embryo development.
Besides that, we observed that addition of NAC during IVM can prevent the compromised
hatching rate promoted by MEFF.
We observed that FF from ME women added to IVM medium of bovine oocytes did not alter
cleavage and blastocyst formation rates, but decreased hatching rate. We expected
to find impairment of cleavage in the group with MEFF due to damages on the meiotic
spindle of oocytes.[14 ]
[15 ]
[28 ] In vivo, cleavage occurs during embryo transit between the oviduct and the uterus,
and many factors can potentially interfere with this process.[29 ] A study using time-lapse showed that meiotic spindle visualization of oocytes is
not related to the morphokinetic of in vitro embryo development, but it could be related
to clinical pregnancy and live births in women with polycystic ovarian syndrome.[30 ] A recent study, assessing the impact of endometriosis on embryo development and
quality after ICSI, showed similar cleavage rates in groups of women with and without
endometriosis, concordant with our findings.[31 ] In another recent study, Sanchez et al.[31 ] did not find differences in the blastocyst formation rate comparing control and
endometriosis groups undergoing ART. Thus, we hypothesize that mild endometriosis
may compromise oocyte quality,[14 ]
[15 ]
[32 ] without interfering with cleavage and blastocyst rates, but reducing hatching rate,
which could explain lower natural fertility in some of these women.[8 ]
[9 ]
Corroborating our findings, Piromlertamorn et al.[33 ] assessed the impact of incubation of mouse oocytes with endometriotic fluid on early
embryo in vitro development, and they also did not show differences in cleavage and
blastocyst formation, but they observed that endometriotic fluid impaired the hatching
rate.[33 ] Interestingly, a randomized clinical trial[34 ] assessing assisted hatching in embryos from women with endometriosis reported higher
implantation and clinical pregnancy rates in the group of women whose embryos had
undergone laser-assisted hatching after ICSI, suggesting that compromised hatching
rate may be involved in lower implantation rates in women with endometriosis. A study,
assessing embryo quality and implantation rate in infertile women undergoing ART,
showed no differences in the number of good quality embryos between women with and
without endometriosis, but reported a statistically significant decrease in implantation
rate in the endometriosis group.[35 ] So, we question whether our findings could explain, at least in part, implantation
failure in women with endometriosis and repeated implantation failure after IVF and
embryo transfer, which needs further investigation.
Hatching is a prerequisite for embryo implantation in the endometrium and depends
on continuous expansion of the blastocele and thinning and rupture of the zona pellucida.[36 ] Goud et al.[37 ] reported a correlation between follicular levels of nitrate (an oxidative end product
of nitric oxide) and zona pellucida dissolution time (an indirect marker of thickness
of the zona pellucida) of oocytes from a group of women with endometriosis. So, we
hypothesize that FF from women with ME, due to OS, may cause alterations on the zona
pellucida of the oocyte,[38 ] which compromises the normal hatching processes.
In relation to the addition of antioxidants, NAC and/or LC did not alter cleavage
and blastocyst rates in groups with FF from ME women and only NAC prevented reduced
blastocyst hatching rates. Thus, we hypothesize that NAC supplementation prevented
OS damages in meiotic spindle and on zona pellucida. N-acetyl-cysteine may also have
reduced the disulfide bonds in the zona pellucida and induced expansion of the zona
pellucida[39 ] culminating in hatching. However, a study using a rat model demonstrated that intravenous
NAC in high concentration (1000 mg.kg-1/day) promotes infertility probably due to
an exacerbated thinning of the zona pellucida.[40 ] Thereby, further studies using animal models are needed to evaluate the effect of
different concentrations of NAC to determine its efficacy and safety for embryos before
its effects can be evaluated in infertile women with endometriosis.
Interestingly, but corroborating our previous study,[15 ] concomitant addition of NAC and LC had equal or inferior results relative to NAC
or LC alone. We can propose one hypothesis to explain this finding: an interaction
of the reactive portions of NAC and LC may have occurred when these antioxidants were
solubilized together in aqueous medium, thereby reducing the efficacy of the clearance
of free radicals.
Using bovine oocytes and FF from ME women, we aimed to mimetize what could happen
in the follicular microenvironment of women with mild endometriosis in natural cycles.
However, data obtained from studies using animal models cannot necessarily be extrapolated
to humans, and studies evaluating in vitro development of embryos from ME women undergoing
ART would be important to confirm our findings. On the other hand, it is important
to state that FF obtained from stimulated cycles not necessarily can represent FF
from a natural cycle, which needs further investigation.
Therefore, FF from infertile women with ME added to the medium of IVM of bovine oocytes
did not interfere with cleavage and blastocyst rates, but impaired hatching rate.
N-acetyl-cysteine prevented these damages, suggesting involvement of OS in the worst
of oocyte and embryo quality of women with ME. Further studies evaluating the potential
clinical application of our findings are needed, especially in terms of improving
natural fertility and/or implantation rates in ME women with recurrent implantation
failure.
