Effects of melatonin and human follicular fluid supplementation of in vitro maturation medium on mouse vitrified germinal vesicle oocytes: A laboratory study

Abstract Background Vitrification as the most efficient method of cryopreservation, enables successful storage of oocytes for couples who undergo specific procedures including surgery and chemotherapy. However, the efficacy of in vitro maturation (IVM) methods with vitrified germinal vesicle (GV) oocytes could be improved. Objective As melatonin and follicular fluid (FF) might enhance IVM conditions, we used these supplements to assess the maturation rate of vitrified GV oocytes and their artificial fertilization rate. Materials and Methods Four hundred mouse GV oocytes were harvested, vitrified, and assigned into control (C-Vit-GV) and treatment groups of melatonin (M-Vit-GV), human follicular fluid (HFF-Vit-GV), and a combination (M + HFF-Vit-GV). A non-vitrified group of GV oocytes (non-Vit-GV) and a group of in vivo matured metaphase II (Vivo-MII) oocytes served as control groups to evaluate the vitrification and IVM conditions, respectively. Maturation of GV oocytes to MII and further development to two-cell-stage embryos were determined in the different groups. Results Development to two-cell embryos was comparable between the Vivo-MII and non-Vit-GV groups. IVM and in vitro fertilization (IVF) results in the non-Vit-GV group were also comparable with the C-Vit-GV oocytes. In addition, the IVM and IVF outcomes were similar across the different treatment groups including the M-Vit-GV, HFF-Vit-GV, M + HFF-Vit-GV, and C-Vit-GV oocytes. Conclusion Employing an appropriate technique of vitrification followed by suitable IVM conditions can lead to reasonable IVF outcomes which may not benefit from extra supplementations. However, whether utilizing other supplementation formulas could improve the outcome requires further investigation.


Introduction
Obtaining qualified oocytes from ovarian stimulation procedures has remained a challenge in common assisted reproductive technologies (ART) (1).
Some harvested immature oocytes may not respond to in vivo gonadotropin stimulus and ART programs, but many are still capable of undergoing spontaneous in vitro maturation (IVM) and in vitro fertilization (IVF) in suitable culture conditions; therefore, the IVM technique was introduced (2). Cryopreservation of immature oocytes and IVM may preserve fecundity in some cases including ovarian cancer. Vitrification, which has the lowest rate of cryoinjuries, is considered the most appropriate method Follicular fluid (FF) contains various hormones, growth factors, glycosaminoglycans, and steroids (5). It is also rich in components crucial to oocyte maturation and embryo development including nutrients, gonadotropins, apoptosis inhibitors, meiosis-activating sterol, etc. IVM outcomes with autologous or heterologous FF are not consistent, and depend on the size of the follicles, the FF resource, and the concentration of FF components (6).
Melatonin is an indolamine derived from the amino acid tryptophan. The pineal gland is the main source of melatonin production, but it is also secreted in other parts of the body such as in the lacrimal gland, gastrointestinal tract, skin, ovary, testes, and liver. This indolamine is responsible for controlling the circadian rhythm and seasonal reproductive regulation; however, recent findings suggest melatonin also has an immunomodulatory function and cytoprotective role. As an antioxidant, melatonin induces DNA repair mechanisms, and regulates the action of other antioxidant agents and cell metabolism (7). Melatonin also plays a role in ovarian function and female reproduction.
Supplementation of the culture medium with melatonin increases the rate of fertilization and enhances the earlier stages of embryonic development (8). Embryo culture medium supplemented with melatonin has improved blastocyst formation, the hatched blastocyst rate and the total blastomere number (9). In humans, melatonin could alter oxidative stress in FF and improve fertilization outcomes following oral consumption (10). Indolamine receptors have been detected in cumulus cells and oocytes (11), suggesting they have a role in oocyte development. Some in vitro studies have also demonstrated positive effects of melatonin use in oocyte maturation protocols and embryo culture. However, the use of melatonin in IVM may not affect (12), inhibit (13), or enhance nuclear maturation (11), and may also have no beneficial effect on embryo development (12).
Considering the controversial impact of FF and melatonin, the aim of the present study was to determine whether supplementation of IVM culture medium with a defined concentration of exogenous melatonin and FF could have any effect on nuclear and cytoplasmic maturation of vitrified oocytes.

Animals
Thirty 6-8-wk-old female NMRI mice were maintained in a 12-hr light/dark cycle at 22-24°C with unrestricted access to food and tap water.

Chemicals
All materials used in the study were purchased from Sigma Chemical Company (St Louis, MO, USA) unless stated otherwise.

GV and MII oocyte collection
In order to collect GV oocytes, the animals were sacrificed, and the ovaries were removed and immediately placed in an α-MEM culture

GV oocyte vitrification and warming
GV oocytes were exposed to an equilibration

Oocyte viability assessment and IVM
After incubation of the oocytes for one hr in 5% CO 2 in humidified air at 37°C, the oocytes were assessed for viability and the ones with

Artificial oocyte activation
In order to determine the effects of melatonin and HFF treatments on the fertilization capacity of in vitro matured MII oocytes, the GV oocytes were transferred to 10 µmol of ionomycin and 2 mmol of 6-dimethyl-aminopurine in α-MEM supplemented with 5% human serum for six min and three hr, respectively. The MII oocytes were then transferred to α-MEM with 20% human serum and assessed for the development to two-cell-stage embryos 24 and 48 hr later using an inverted phase-contrast microscope (Olympus, IX71, Japan).

Ethical considerations
The experiments and animal use were

Results
The outcomes related to the effect of melatonin, HFF, and their combination on IVM (development to GVBD and MII) and IVF (development to two-cell embryo) of 500 vitrified mouse oocytes are presented below.

Competence of the IVM medium
The developmental rate of non-Vit-GV and Vivo-MII oocytes to two-cell embryos was compared to assess the competence of the IVM conditions. Following artificial oocyte activation,

Oocyte activation and development to two-cell embryos
Observing the pronucleus in any of the matured oocytes (MII) was considered the initial sign of oocyte activation to undergo development to the two-cell stage. Data shown in Figure 1 suggest that  with non-vitrified human oocytes (14). However, no differences in the incidence of diploid metaphase II bovine oocytes among control, vitrified, and non-vitrified groups has been detected (15).
Similar survival rate in both mature and immature vitrified oocytes after warming was also reported.
However, they found a significantly higher oocyte maturation rate in the oocytes that had undergone IVM before vitrification than in the oocytes vitrified after IVM (16). Our results also revealed that the employed IVM protocol could support the maturation of vitrified GV oocytes as no significant difference was found regarding the competence to undergo GVBD and MII stage.
FF has a crucial role in nutritional and hormonal support of oocytes particularly before ovulation (17). However, findings from other studies regarding the positive impact of FF on in vitro conditions are inconsistent. When GV oocytes were cultured in pure FF derived from small follicles 1-5 mm in diameter, the maturation rate was significantly low (18). In contrast, a higher rate of pronucleus formation was detected when the culture medium was enriched with porcine FF (19). Other studies have also reported that supplementation of maturation medium with 10% FF obtained from small, medium, large, and pre-ovulatory follicles (18), and also 20% FF (20) improved

Conclusion
It can be concluded that GV oocyte vitrification