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 Table of Contents  
ORIGINAL ARTICLE
Year : 2019  |  Volume : 3  |  Issue : 1  |  Page : 42-48

Preliminary research on the effect of linolenic acid on human oocyte maturation


1 Reproductive Medical Center, The Third Affiliated Hospital of Zhengzhou University, Zhengzhou 450052, Henan, China
2 Medical College of Pingdingshan University, Pingdingshan 467000, Henan, China

Date of Submission11-Sep-2018
Date of Web Publication11-Apr-2019

Correspondence Address:
Li-Jun Sun
Reproductive Medical Center, The Third Affiliated Hospital of Zhengzhou University, 7 Kangfu Forestreet, Erqi District, Zhengzhou 450052
China
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/2096-2924.255988

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  Abstract 


Objective: To investigate the effects of exogenous α-linolenic acid (ALA) on in vitro maturation (IVM) and developmental competence of human oocytes.
Methods: Experiment 1 examined the effects of ALA at different concentrations (0 [control], 10, 50, 100, and 200 μmol/L) in the IVM medium on oocyte maturation. Treatment with 50 μmol/L ALA significantly accelerated oocyte maturation (P < 0.05) and resulted in significantly higher mitochondrial DNA (mtDNA) copy number compared to the control. Hence, 50 μmol/L ALA was selected for combination with follicular fluid (FF) to investigate oocyte developmental potential. mtDNA of the matured oocyte was analyzed by real-time polymerase chain reaction. Experiment 2 investigated the effects of FF with optimal ALA concentration (Group A: ALA + FF + IVM medium) or without ALA (Group B: FF + IVM medium) on oocyte maturation, fertilization, 2 pronuclear cleavage, and embryo and blastocyst development. Malondialdehyde (MDA) content and superoxide dismutase (SOD) activity were measured for maturation medium from Group A, Group B, and Group C (control group, IVM medium only).
Results: Treatment with 50 μmol/L ALA obviously accelerated oocyte maturation (P < 0.05) and resulted in significantly higher mtDNA copy number (P < 0.05) in the matured oocytes compared to the control (0 μmol/L ALA). Supplementation of 50 μmol/L ALA and FF (Group A) obviously increased the total maturation rate than FF-treated group (Group B) which has higher (P < 0.05) total maturation rate than that of Group C. However, no significant differences were observed in fertilization, embryo availability, and blastocyst production among Group A, B, and C. Treatment with 50 μmol/L ALA decreased the level of MDA (P < 0.05), but had no effect on the activity of SOD in the IVM medium.
Conclusions: Our results suggested that the treatment with 50 μmol/L ALA during IVM improves maturation in human oocytes. It is also likely to improve embryo availability and blastocyst formation. This might be associated with the alteration of mtDNA replication (increased mtDNA copy number) and the reduction of oxidative stress (reduced MDA level).

Keywords: Human Oocyte; In vitro Maturation; Mitochondrial DNA; Oxidative Stress; α-Linolenic Acid


How to cite this article:
Hu JJ, Li JX, Wang XL, Guan YC, Sun LJ. Preliminary research on the effect of linolenic acid on human oocyte maturation. Reprod Dev Med 2019;3:42-8

How to cite this URL:
Hu JJ, Li JX, Wang XL, Guan YC, Sun LJ. Preliminary research on the effect of linolenic acid on human oocyte maturation. Reprod Dev Med [serial online] 2019 [cited 2019 Apr 24];3:42-8. Available from: http://www.repdevmed.org/text.asp?2019/3/1/42/255988




  Introduction Top


Dietary intake of different types of polyunsaturated fatty acids (PUFAs) can influence fertility in animals and humans.[1] Immature oocytes must undergo both nuclear and cytoplasmic changes that are essential for fertilization and development.[2] Oocytes acquire developmental competence during growth and maturation. These effects may partly be mediated by a direct action of fatty acids on oocyte development in vivo or in vitro. A dose–response study shown that supplementation of 50 μmol/L α-linolenic acid (ALA) to bovine oocytes during maturation improves oocyte developmental potential.[3] They found the importance of prostaglandin (PG) synthesis and the mitogen-activated protein kinase pathway in mediating the effect of ALA on oocyte maturation.

Mitochondrial function plays an important role in oocyte maturation and subsequent embryonic development.[4] A study has previously shown that mitochondrial DNA (mtDNA) copy number plays a critical role in oocyte maturation and subsequent embryonic development, rendering it an important marker of oocyte quality.[5] However, the effect of mtDNA on oocyte maturation and ensuing embryonic development remains inconclusive.

Studies have shown that the level of internal oxidative stress (OS) increases as a function of aging, especially for women in their reproductive years.[6] Mitochondrial dysfunction in aged oocytes results in an increase in reactive oxygen species (ROS) and decrease in adenosine 5'-triphosphate.[7] Although a little ROS exists in medium during normal physiological activity, the continued elevation of ROS in cells can cause detrimental effects on mitochondria, DNA, proteins, and lipids.[8]

Oocytes cultured in vitro are inevitably exposed to more oxygen than that in vivo; the excessive oxygen radicals generated in the culture medium can compromise oocyte maturation and embryonic development. Hence, it is important to overcome OS and reduce the damage of free radicals to the cell with antioxidant supplementation.

Linolenic acid (18:3 [n-3]), or all-cis 9, 12, 15-octadecatrienoic acid, is an n-3 PUFA. It is an important component of cell membranes and acts as a precursor to eicosapentaenoic acid and dehydroacetic acid. ALA not only increases the levels of PGE2 and cyclic adenosine monophosphate in oocytes but also inhibits the peroxidation reaction and the creation of malondialdehyde (MDA) by enhancing the vitality of antioxidases, such as superoxide dismutase (SOD) and glutathione peroxidase (GPx), which contribute to oocyte maturation. The oxidation of unsaturated fatty acids enhance mitochondrial functions and improve oocyte maturation and cleavage in pigs.[9] ALA supplementation in vitro maturation (IVM) had no effect on mitochondrial distribution and activity, decreased ROS levels compared with the control, and was associated with an increased nuclear maturation rate. This may be due to an effect of ALA on the expression of antioxidant enzymes leading to no accumulation of ROS during maturation time.[10] Some studies have revealed that ALA had an effect on mammalian oocyte maturation in vitro, but this effect was not previously reported in human oocyte maturation in vitro until now.[3] According to our previous study, the addition of mature follicular fluid (FF) to the IVM medium enhanced the rates of maturation, fertilization, and cleavage more effectively than the addition of gonadotropin (Gn). In the present study, we add mature FF based on a predetermined optimal ALA concentration to explore the effect of ALA on human oocyte maturation and subsequent embryonic development.


  Methods Top


Materials

Except when stated otherwise, all chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA). All solutions were prepared using reagent-grade chemicals and deionized-distilled water. IVM media were incubated overnight at 37°C under a humidified atmosphere of 5% CO2 before use in culture.

Patients

From October 2014 to October 2015, 423 infertile women whose husbands were sterile (presenting oligoasthenozoospermia, oligoasthenospermia, teratozoospermia, and/or azoospermia) were enrolled for this study. The patients were divided into two groups. Group 1 contained 273 patients with a mean age of 30.6 ± 4.0 years, from which 273 metaphase I (MI)-stage oocytes and 262 germinal vesicle (GV)-stage oocytes were obtained. According to [Table S1], these patients were separated into five sections with no obvious differences in age, duration of infertility, body mass index (BMI), and endocrine basis (bFSH, bE2, and bLH) (P > 0.05). Group 2 contained 150 patients with a mean age of 31.2 ± 6.0 years, from which 87 MI-stage oocytes and 161 GV-stage oocytes were collected. As shown in [Table S2], these patients were separated into three sections with no obvious differences in age, duration of infertility, BMI, and endocrine basis (bFSH, bE2, and bLH) (P > 0.05). Only healthy sperm (density >15 × 106/mL, progressive rate [PR] >32%, sperm with normal morphology rate >4%) collected from patients undergoing in vitro fertilization and embryo transfer (IVF-ET) was used for IVF. This study was approved by the ethics committee and all patients signed informed consent.



Experimental design

Experiment 1 – The immature oocytes collected from Group 1 were randomly divided into five sections according to equiproportional MI and GV oocytes. Then, they were incubated with different concentrations of ALA (0 [control], 10, 50, 100, and 200 μmol/L) in basal IVM medium. After 24 h in culture, the maturity of the oocytes was assessed using an inverted microscope (CKX41, Olympus, Tokyo, and Japan), and the matured oocytes were collected for mtDNA copy number analysis.

Experiment 2 – The immature oocytes harvested from Group 2 were randomly separated into three sections according to equiproportional MI and GV oocytes. Later, three sections of immature oocytes were treated with different medium constituents (Group A, 50 μmol/L ALA + 50% FF + 50% basal IVM medium; Group B, 50% FF + 50% basal IVM medium; and Group C, basal IVM medium only) to assess the effects of ALA on oocyte maturation, fertilization, 2 pronuclear (2PN) cleavage, and embryo and blastocyst development. The maturation medium was collected and frozen for future measurements of MDA content and SOD activity.

Oocyte collection

All patients were given a daily subcutaneous injection of 0.05 mg Gn-releasing hormone analog (Ipsen Pharma Biotech, Signes, France) from the midluteal phase of the preceding cycle until the average diameter of the leading follicle reached 18–20 mm (or two follicles with a mean diameter ≥17 mm, three follicles with a mean diameter ≥16 mm), with patients receiving varying doses of Gn according to the size and number of follicles that developed and their E2 levels. Then, intramuscular human chorionic gonadotropin (hCG) at doses of 5,000–10,000 IU was administered or recombinant hCG at 250 μg/L was triggered. Under transvaginal ultrasound guidance, oocyte retrieval was performed 34–36 h after hCG injection. According to their morphology, the collected oocytes were classified into three stages: GV, MI, and MII. The oocytes at the GV and MI stages were selected for the IVM procedure.

In vitro maturation

The collected immature oocytes were transferred to basal IVM medium for culturing according to the experimental design. For 10 mL basal IVM medium preparation, 100 μL of 10 mM ALA and 50 μL penicillin and 50 μL streptomycin were added to 9.8 mL G-IVF+. G-IVF+ was made of 9 mL G-IVF (Vitrolife AB, Gothenburg, Sweden), 1 mL fatty acid-free human serum albumin (Vitrolife AB), and 75 IU human menopausal gonadotropin (75 IU FSH and 75 IU LH). The IVM medium should be freshly made the day before oocyte retrieval and incubated overnight at 37°C under a humidified atmosphere of 5% CO2 before use in culture. The immature oocytes were incubated for 24 h at 37°C in 5% CO2 in an incubator (K-SYSTEMS, Birkeroed, Denmark) with high humidity.

Oocyte maturation assessment and mature oocyte storage

Oocyte maturation was examined for the presence of the first polar body under an inverted microscope. The number of mature oocytes (MII) was recorded for maturation rate calculation. The MII oocytes were collected and washed three times with phosphate-buffered saline and then a single MII oocyte was transferred to 10 μL cell lysis buffer (50 mmol/L Tris-HCl, 0.1 mmol/L EDTA, 0.5% Tween-20, 200 mg/mL protease K, pH 8.5) (Promega, Madison, WI, USA) in a 0.2 mL thin-walled polymerase chain reaction (PCR) tube for total DNA extraction.

Measurement of the mitochondrial DNA copy number

The mtDNA copy number in MII oocytes collected from the 0 μmol/L ALA (control) group and 50 μmol/L ALA group was examined in this study. MII oocytes stored in 0.2 mL PCR tubes were lysed at 55°C for 2 h and then treated with protease K denatured at 95°C for 5 min; this crude lysate was used as template DNA. The mtDNA copy number was then determined by real-time PCR using a real-time rotary analyzer (Eppendorf Mastercycler® 5333, Hamburg, Germany). The PCR reaction was assembled as follows: 2.5 μL of template DNA (crude lysate or standard curve plasmid dilution), 0.4 μL of each mtDNA target-specific primer set (10 μmol/L, forward primer 5'-CGAAAGGACAAGAGAAATAAGG-3' and reverse primer 5'-CTGTAAAGTTTTAAGTTTTATGCG-3'), 10 μL SYBR Green PCR Master Mix (Takara, Dalian, China), and 6.7 μL H2O was combined in each well of a 96-well PCR plate. Each sample was amplified in triplicate and the data were averaged. The PCR was performed with initial denaturation at 95°C for 5 min, followed by 40 cycles of 95°C for 15 s, 60°C for 15 s, and 72°C for 20 s. The melting curve was calculated for each sample to ensure the formation of a single PCR product. A standard curve was generated for each run using 10-fold serial dilutions of mtDNA copy number standard curve plasmid. For construction of the standard curve plasmid, PCR was performed with the primer set described above to amplify a 158 bp fragment and then the DNA fragment was cloned into a pMD18-T vector (Takara). The plasmid was sequenced for the confirmation before use. The copy number for each sample was calculated as follows: the mtDNA cycle threshold (CT) values were averaged for three technical repeats and then the average CT values of the sample were compared with the average CT values of the standard curve to determine the copy number per PCR reaction.

Follicular fluid collection

We collected the FF when selecting the biggest follicle (diameter ≥16 mm) under B-ultrasound guidance. The fluid was cleared by centrifugation at 25°C for 20 min at 600 ×g. The supernatant was transferred into a clean tube and incubated at 53°C for 30 min, followed by filtration through a 0.22-μm filter and storage at −20°C.

Sperm collection and preparation

Four milliliters of healthy sperm (density >15 × 106/mL, PR >32%, sperm with normal morphology rate >4%) was collected from patients undergoing IVF-ET for IVF. The sperm was incubated at 37°C until melting was complete, and then mixed well with equal volume of cryoprotective medium for human spermatozoa. Then, 30 μL sperm mixture was pipetted into 2-mL tubes and frozen above liquid nitrogen for 1 min. Later, the tubes were immersed in liquid nitrogen for storage. Before performing IVF, the sperm mixture was removed from the liquid nitrogen and thawed in a 37°C water bath. The recovered sperm mixture was washed upon transfer to a tube containing 2 mL IVF buffer, followed by centrifugation for 15 min at 500 × g; the supernatant was then removed. The wash step was repeated, after which the density of sperm was adjusted to (1–10) × l05/mL by adding 0.2 mL IVF+ buffer. The sperm was then incubated at 37°C in 5% CO2 in an incubator (K-SYSTEMS, Birkeroed, Denmark).

In vitro fertilization

The sperm suspension was transferred to the polyvinylpyrrolidone (PVP) droplet in a microinjection dish and allowed to spread out to periphery of the droplet for several minutes. The microinjection dish was placed on the heating board of the micromanipulator, with the injection capillary positioned downward to absorb the small amount of PVP. The capillary was then placed over 1/3 of the sperm tail to aspirate the sperm head. The stage was moved to the injection droplet, which was aspirated to hold the oocyte using the holding capillary, placing the polar body at the 6 or 12 o'clock position. The oolemma was pushed quickly by the injection capillary until the relaxation of the oolemma was observed, advancing the capillary until it reached 3/4 of the oocytes, and then the sperm was injected. The capillary was then withdrawn gently.

Assessment of nuclear stage of zygotes and embryo quality

After injection, the oocytes were transferred to G-1+ medium and incubated at 37°C in 5% CO2 in an incubator (K-SYSTEMS, Birkeroed, Denmark). Fertilization was assessed at 16–20 h after IVF by determining the presence or absence of pronuclei. Lack of a pronucleus indicated that fertilization had been aborted. The presence of two pronuclei (male and female) was counted as a 2PN stage or normal fertilization. Only one or more than 2PN represent asynchronous fertilization. The fertilized zygotes were further cultured in the same medium. The embryo quality was evaluated at 68–72 h after IVF, based on the scoring criteria established by our reproductive center. The embryos were scored as follows: Grade I, blastomeres of equal size with ≤5% fragmentation, without multinucleation; Grade II, blastomeres of slightly unequal size and <6%–20% fragmentation, without multinucleation; Grade III, blastomeres of unequal size with <21%–50% fragmentation, without multinucleation; and Grade IV, blastomeres of equal and unequal size with ≥50% fragmentation, with multinucleation. Grade I and Grade II embryos were defined as excellent-quality embryos, Grade III embryos were defined as available embryos, and Grade IV embryos were generally excluded from transfer.

Assessment of blastocysts

The embryos were transferred to G-2+ medium for further incubation in a 5% O2, 6% CO2 incubator (William A. COOK Australia Pty. Ltd.) at 37°C. After 48–72 h in culture, the morphological structures of embryos were examined to assess blastocyst formation. Approximately 5 days after IVF, the embryo reached the blastocyst stage, whereupon the cells in the embryo were differentiated to form the outer layer called the trophoblast (TE) and the inner cell mass (ICM). The blastocyst quality and blastocyst expansion grade were evaluated using the scoring system developed by Dr. David Gardner.[11] Briefly, ICM was classified by the following descriptions: (A) tightly packed with many cells, (B) loosely grouped with several cells, and (C) very few ICM cells. TE was classified into three categories: (A) many cells forming a cohesive layer, (B) few cells consisting of a loose epithelium layer, and (C) very few large cells.

Measurement of malondialdehyde and superoxide dismutase activity

After the in vitro matured oocytes were collected to perform IVF, the maturation medium was preserved for the measurement of MDA levels and SOD activity. We measured MDA and SOD following the instructions provided by the manufacturer of the respective reagent kits (Nanjing Jiancheng Biology Engineering Institute, Nanjing, China).

Statistical analysis

Statistical analysis was performed using SPSS 17.0 (SPSS Inc., Chicago, IL, USA). The statistical results of measurement data were expressed as mean ± standard deviation ( ± s); a t-test was used to compare the two groups of independent samples, one-way analysis of variance was used to compare multiple groups when the data followed the normality and homogeneity of variance; the Kruskal–Wallis test was used otherwise. In multiple comparisons, the Bonferroni method was used for comparison between two groups. The level of significance in multiple comparisons was adjusted according to the comparison times (k), α' = α/k (α = 0.05), P values < α' were considered statistically significant.


  Results Top


Experiment 1: Effects of different α-linolenic acid concentrations in maturation medium on oocyte maturation

The effects of treatment with ALA at various concentrations (0, 50, 100, and 200 μmol/LALA) during IVM on the nuclear maturation were examined to determine the optimal concentration. In this experiment, as shown in [Table 1], 24 h after IVM, the 50 μmol/L ALA group significantly increased total maturation rate (70.1%) compared with other ALA treatment groups (42.1%, 51.1%, 56.7%, and 34.1% for 0 [control], 10, 100, and 200 μmol/L, respectively; P < 0.05). When we considered the maturation rate of MI-and GV-stage oocytes, 50 μmol/L ALA also had significant effects (64.4%) on the maturation of GV-stage oocytes compared with various concentrations of ALA (P < 0.05), while the proportion of mature MI-stage oocytes did not differ among the control (61.3%) and the various concentrations of ALA (68.4%, 78.9%, 74.4%, and 60.6% for 10, 50, 100, and 200 μmol/L, respectively). It seemed that GV-stage oocytes were more sensitive to ALA treatment than MI-stage oocytes.
Table 1: Effects of ALA concentrations (0, 10, 50, 100, and 200 μmol/L) added to IVM medium on GV- and MI-stage oocytes after maturation for 24 h

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Experiment 2: Effects of exogenous α-linolenic acid in maturation medium on oocyte mitochondrial DNA copy number

Based on the results of Experiment 1, we selected 50 μmol/L ALA as the optimal concentration for oocyte maturation. In Experiment 2, matured oocytes from the 0 and 50 μmol/L ALA treatment groups were collected for the measurement of the mtDNA copy number. To investigate the influence of mtDNA on oocyte maturation, we compared the mtDNA content of matured oocytes from the 50 μmol/L ALA group with matured oocytes from the 0 μmol/L ALA group. As shown in [Table 2], the average mtDNA copy number of the 50 μmol/L ALA group, 3.64 × 107 (±1.85 × 106), was significantly different (P < 0.05) from that of the 0 μmol/L ALA group, 3.42 × 106 (±1.95 × 105).
Table 2: The effects of 50 μmol/L ALA on mtDNA copy number

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Experiment 3: Effects of α-linolenic acid and follicular fluid in maturation medium on oocyte maturation, fertilization, 2 pronuclear cleavage, and embryo and blastocyst development

Based on the results of the previous experiments, the 50 μmol/L ALA concentration was selected for combination with FF for further evaluation of oocyte maturation and developmental potential after IVF. As shown in [Table 3], the total maturation rate was higher (P < 0.05) in the 50 μmol/L ALA and FF-treated group (75.9%, Group A) compared to the FF-treated group (57.3%, Group B). The FF-treated group exhibited an increased maturation rate (P < 0.05) compared to the control group (38.0%, Group C). These data revealed that both ALA and FF promote oocyte maturation. When we examined the maturation rate of MI-stage oocytes among the three groups, we found that only Group A (83.9%) showed higher (P < 0.05) MII rate than Group C (53.6%). The maturation rate of GV, Group A (71.4%) was significantly higher (P < 0.05) than that of Group B (46.3%) and Group C (29.4%). As shown in [Table 4], 66, 47, and 30 MII stage oocytes were collected from Group A, B, and C, respectively. After IVF, although the fertilization rates were similar among each group (P > 0.05), oocytes from Group A (92.3%) and Group B (88.6%) had a significantly higher cleavage rate than oocytes from Group C (57.9%). However, no significant differences were observed in embryo availability and blastocyst production among the three groups.
Table 3: The maturation rate of oocytes treated in three different in vitro maturation medium after 24 h

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Table 4: The embryonic development of oocytes maturated in three different IVM medium after IVF

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Experiment 4: Effects of exogenous α-linolenic acid in maturation medium on superoxide dismutase activity and malondialdehyde level

After the MII-stage oocytes were collected to perform IVF, we preserved the maturation medium from Groups A, B, and C to measure the activity of SOD and the level of MDA. As shown in [Table 5], ALA did not alter the activity of SOD, since the activity of SOD was similar among Group A (ALA + FF, 1.71 U/mL ± 0.05), Group B (FF, 1.72 U/mL ± 0.04), and Group C (control, 1.70 U/mL ± 0.06) (P > 0.05). However, the level of MDA in Group A (42.50 nmol/mL ± 9.79) was lower (P < 0.05) than that in Group B (47.31 nmol/mL ± 7.90) and Group C (49.28 nmol/mL ± 7.82). This result suggested that other antioxidant species might be responsible for the decrease in MDA levels.
Table 5: The activity of SOD and content of MDA in three different IVM media after 24 h

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  Discussion Top


During processing of IVM oocytes, the provision of suitable IVM media could improve their developmental potential. Many studies in animals have shown that addition of ALA to IVM medium could promote oocyte maturation and even improve subsequent embryonic development.[10],[12],[13] This study aimed to investigate the effects of supplementary ALA on human oocyte maturation and development in vitro. According to the previous studies, the concentrations of ALA in FF were 0.018 mg/mL (64.6 μmol/L) and 0.028 mg/mL (100.6 μmol/L) for small and large follicles from sheep, respectively. The concentrations of ALA in cattle serum were 0.005 mg/mL (53.9 μmol/L) and 0.04 mg/mL (143.66 μmol/L).[14],[15] Hence, to mimic the normal physiological situation in vitro, a range of 0, 10, 50, 100, and 200 μmol/L ALA was added to IVM medium to investigate the effects of supplementary ALA on human oocyte maturation and development.

Our results showed that treatment of GV-stage oocytes with 50 μmol/L ALA resulted in the highest maturation rate (MII-stage) compared to the other concentrations tested (0, 10, 100, and 200 μmol/L ALA). However, ALA at high concentration (200 μmol/L) reduced the oocyte maturation. The observations were similar to a previous study with bovine oocytes reported,[3] which elucidated that ALA at 50 μmol/L significantly increased the percentage of oocytes at the MII-stage, while high concentration ALA could be harmful for the bovine oocytes. Interestingly, our results revealed that ALA did not affect MI-stage oocyte maturation, but ALA at 50 μmol/L significantly improved GV-stage oocyte maturation. It seemed that the addition of ALA in IVM medium benefited GV-stage oocyte more than MI-stage oocyte. This result is kind of supported by the evidence that ALA is involved in the regulation meiotic arrest at the GV stage and in preventing GV breakdown.[16],[17]

Usually, the immature oocytes are retrieved from small follicles; hence, they are deficient in the vital maturation factors in the cytoplasm and are not exposed to the hormonal microenvironment of ovulatory follicles.[18],[19] The provision of suitable IVM medium could help immature oocytes improve their subsequent developmental potential. Our previous studies found that the addition of FF to IVM medium could promote human oocyte maturation, fertilization, and cleavage (data not shown). This was in agreement with the published results in which indicated that the culturing of oocytes in FF benefits cumulus expansion and nuclear maturation, enabling the oocytes to develop into the blastocyst stage.[20] In this study, we found that the oocytes cultured in Group B (FF + IVM medium) had a significantly higher total maturation rate and cleavage rate than those in Group C (IVM medium). This result was quite consistent with our previous result. However, when we specifically look at the MII rate of GV-stage oocytes, no significant difference was observed between Group B (FF + IVM medium) and Group C (IVM medium). On the contrary, FF did promote the MI-stage oocytes maturation. Compared to the FF group (Group B), we only observed a higher MII rate when the GV stage oocytes were treated with the ALA and FF combination medium (Group A). These results suggest that FF benefits MI-stage IVM oocytes, while ALA has a much greater effect on GV-stage IVM oocytes. Moreover, we found a higher available embryo rate and blastocyst formation rate in the Group A (ALA + FF + IVM medium) and Group B (FF + IVM medium) compared to the control group; however, statistical analysis showed no significant difference between the three groups. This is likely due to sample size restriction. An effective statistical analysis could not be performed given the formation of only one blastocyst in the control group.

It is known that both nuclear and cytoplasmic maturation play an important role in determining the quality and the developmental competence of oocytes. Here, we tried to discuss the effect of ALA on the human oocyte maturation and further development from cytoplasmic maturation. Studies on mammalian oocytes suggested that the number of mitochondria is one of the important markers of cytoplasmic maturation, which could be represented by mtDNA copy number (each oocyte mitochondrion has only one copy of its genome), is strongly linked with the quality of oocytes and developmental competence.[5],[21],[22],[23] A study on mice found that mature oocytes have a higher mtDNA copy number than immature oocytes.[24] In the present study, we identified that mtDNA copy number in the oocytes treated with 50 μmol/L ALA was significantly higher than the oocytes in the control group (0 μmol/L ALA), which is coincident with the oocyte maturation rate. This result suggested that ALA treatment increased oocyte mtDNA copy number, resulting in improvement of oocyte quality. Ge et al. reported that the inhibition of mitochondrial metabolic activity during oocyte maturation has a negative impact on both oocyte maturation and subsequent embryonic development potential.[4] Previous studies on the function of mtDNA focused mainly on animal models; therefore, whether the mtDNA copy number in human oocytes has the same impact on oocyte IVM and subsequent embryonic development potential requires further study.

The FF microenvironment plays a crucial role in determining the oocyte quality and the embryo quality. OS in FF is usually associated with decreased oocyte developmental potential.[25] A previous study found that omega-3 PUFA supplementation attenuates OS and inflammation in animals, probably through the inhibition of lipid peroxidation.[26] ALA is an essential omega-3 PUFA that contains several olefinic bonds, which could prevent lipid peroxidation by enhancing the activity of antioxidants such as SOD and GPx to clean free radicals and reduce the production of MDA.[12] Hughes et al. have shown that with omega-3 PUFA-containing serum supplementation in the synthetic oviduct fluid medium, the blastocyst formation rate was increased while the expression of SOD was increased. In the present study, we measured the activity of SOD and the level of MD for each group (A, B, and C) of maturation medium.[13] We found that the level of MDA in Group A (ALA + FF) was obviously lower than that of Group B (FF) and Group C (control). This observation combined with the findings that oocytes cultured in the Group A media had a significantly higher oocyte maturation rate than the other two groups, suggesting that oocyte maturation is probably related to ALA-mediated OS reduction. However, we found that the reduction of MDA did not depend on the activity of SOD, since the activity of SOD was similar among the three groups of IVM medium (Group A, Group B, and Group C). It is possible that ALA regulates the OS through other antioxidants besides SOD, such as GPx, glutathione (GSH), catalase (CAT), and Fe3+. So far, due to the limitation of sample size and experimental conditions, the level of GPx, CAT, and Fe3+ in the maturation medium was not determined in this study; therefore, the mechanism by which ALA reduces OS and by which antioxidants remains unclear. Further research in this direction will be important for further developments in IVM technology.

In conclusion, the present study revealed that supplementation of IVM medium with 50 μmol/L ALA improved human oocyte maturation rate in vitro, with the effect of ALA on the maturation of GV-stage oocytes being more obvious than the effect on MI-stage oocytes. It seems that ALA promotes human oocyte maturation and subsequent embryonic development by increasing the oocyte mtDNA copy number and reducing OS.

Supplementary information is linked to the online version of the paper on the Reproductive and Developmental Medicine website.

Financial support and sponsorship

This work has been supported by the Henan Province Medical Science and Technique Project (grant no. 201403109).

Conflicts of interest

There are no conflicts of interest.



 
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  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5]



 

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