Effect of melatonin on the clinical outcome of patients with repeated cycles after failed cycles of in vitro fertilization and intracytoplasmic sperm injection.

To explore whether embryo culture with melatonin (MT) can improve the embryonic development and clinical outcome of patients with repeated cycles after in vitro fertilization/intracytoplasmic sperm injection (IVF/ICSI) failure, immature oocytes from controlled ovarian superovulation cycles were collected for in vitro maturation (IVM) and ICSI. The obtained embryos were cultured in 0, 10-11, 10-9, 10-7 and 10-5 M MT medium respectively, and 10-9 M was screened out as the optimal concentration. Subsequently, 140 patients who underwent failed IVF/ICSI cycles received 140 cycles of embryo culture in vitro with a medium containing 10-9 M MT, these 140 MT culture cycles were designated as the experimental group (10-9 M group), and the control group was the previous failed cycles of patients (0 M group). The results showed that the fertilization, cleavage, high-quality embryo, blastocyst, and high-quality blastocyst rates of the 10-9 M group were significantly higher than those of the 0 M group (P < 0.01; P < 0.01; P < 0.0001; P < 0.0001; P < 0.0001). To date, in total, 50 vitrified-warmed cycle transfers have been performed in the 10-9 M group and the implantation rate, biochemical pregnancy rate and clinical pregnancy rate were significantly higher than those in the 0 M group (all P < 0.0001). Two healthy infants were delivered successfully and the other 18 women who achieved clinical pregnancy also had good examination indexes. Therefore the application of 10-9 M MT to embryo cultures in vitro improved embryonic development in patients with repeated cycles after failed IVF/ICSI cycles and had good clinical outcomes.Trial registration: ChiCTR2100045552.


Background
Assisted reproductive technology (ART) involves the use of medical methods to arti cially manipulate oocytes, sperm, fertilized eggs, and embryos to achieve the goal of conception [1]. Among them, in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) are the core technologies of ART. IVF refers to the natural fertilization of eggs and sperm through arti cial methods and the subsequent in vitro culture of early embryos, while ICSI refers to the process of injecting a single sperm into the cytoplasm of an egg using micromanipulation technology to achieve fertilization. In vitro fertilization and embryo transfer (IVF-ET) technology has become an important means of infertility treatment since its discovery, but the risk of poor embryo development with the traditional IVF/ICSI treatment cycle is still unavoidable [2], which leads to pregnancy failure due to poor development of the embryos transferred. Therefore, exploring an e cient technology of embryo culture in vitro to obtain high-quality embryos is one of the key features for improving the effect of ART treatment.
Melatonin (MT) is a multifunctional molecule that is produced mainly in the pineal gland [3]. It is a powerful free radical scavenger and antioxidant [4]. MT and its metabolites can directly remove reactive oxygen species (ROS) in cells, activate antioxidant enzymes, increase the expression level and activity of glutathione (GSH), and inhibit pro-oxidant enzymes to reduce cell oxidation injury, which can improve oocyte in vitro maturation (IVM) and embryonic development [5]. It has been reported that adding the appropriate amount of MT to IVM and embryo culture medium can signi cantly improve the developmental potential of oocytes and the in vitro development of embryos [6][7][8]. Through research on human IVF and IVM, Li et al. found that the addition of MT to the embryo culture medium can improve the clinical outcomes of IVF and IVM [9]. In 2019, our research team added MT to IVM medium to culture human immature oocytes from a controlled ovarian hyperstimulation (COH) cycle, which signi cantly reduced the levels of ROS and Ca 2+ in oocytes during the IVM process, increased mitochondrial membrane potential, and improved embryo development, eventually resulting in healthy offspring [10]. The present study was the rst attempt to use embryo culture medium supplemented with MT to culture embryos of patients undergoing repeated cycles after failed IVF/ICSI cycles and aimed to explore whether the application of MT could improve the embryonic development and clinical outcomes of patients with repeated IVF/ICSI cycles.

Materials And Methods
Ethics approval and consent to participate This study was approved by the Ethics Committee of Anhui Medical University(2015013). Before conducting the study, the partners of all enrolled patients were interviewed and provided signed informed consent.

Research patients
A total of 140 patients with failed cycles who visited the Reproductive Medicine Center of the First A liated Hospital of Anhui Medical University from June 2020 to November 2020 were selected. The inclusion criteria were as follows: (1) at least one IVF/ICSI failed cycle and (2) patient age ≤36 years. After genetic factors of both men and women were excluded, the causes of infertility included 18 cases of male-only factors, involving oligoasthenoteratozoospermia; 77 cases of female-only factors, including 41 cases of sequelae of pelvic in ammatory disease, 26 cases of polycystic ovary syndrome, and 10 cases of endometriosis; 40 cases of combined male and female factors; and 5 cases with unknown causes.

COH regimen
All patients in this study were treated with gonadotropin hormone-releasing hormone (GnRH) antagonist ( exible regimen) for ovarian stimulation. Recombinant human follicle-stimulating hormone (Gn, Gonal F; Serino Barueri, SP, Brazil) was given on the second or third day of the menstrual cycle for ovarian hyperstimulation. After four to ve days, the dosage of Gn was adjusted according to the follicle size and hormone level. When the dominant follicle reached 12-14 mm, GnRH antagonist (GnRH-A, Cetrotide, Merck Serono SA -Geneva, Switzerland) was added until the day of human chorionic gonadotropin (HCG) injection. When there were two or three follicles with a diameter ≥18 mm, HCG (10,000 U; Pregnyl; AESCA Pharma, Austria) was injected, and oocyte retrieval was completed after 36-38 h under the guidance of a transvaginal ultrasound.

Experiment 1
Immature human oocytes obtained from the COH cycle were collected for IVM to obtain mature oocytes (IVM-MII) in vitro and perform ICSI insemination. Then, embryos were cultured in vitro in medium containing 0, 10 -11 , 10 -9 , 10 -7 or 10 -5 M MT. Ultimately, 10 -9 M was determined to be the optimal concentration of MT.

Experiment 2
For the 140 enrolled patients with failed IVF/ICSI cycles, embryo culture medium containing 10 -9 M MT was prepared to conduct embryo culture in vitro in the subsequent 140 repeated cycles. High-quality blastocysts were collected and cryopreserved. These 140 MT culture cycles were designated as the experimental group (10 -9 M group), and the control group consisted of the previous failed cycles of the same patients (0 M group). In addition, according to the number of failed cycles, the patients were further divided into subgroups of one, two, or at least three failed cycles, and the fertilization and embryo development status of each subgroup in the 10 -9 M and 0 M groups were compared and analysed.

Experiment 3
After 3 months, vitri ed-warmed embryo transfer was performed. The experimental process is shown in Fig. 1.

IVM of oocytes from COH cycles
Human immature oocytes with normal morphology from the COH cycle were collected and placed into IVM medium prepared and balanced overnight to culture for 24 h. Subsequently, the IVM-MII oocytes were selected for ICSI insemination, followed by ve or six days of embryo culture in vitro. The detailed process has been previously reported in published literature [11].
Preparation of the embryo culture medium An appropriate amount of embryo culture medium (cleavage or blastocyst; Cook, Sydney, Australia) was prepared, and then an appropriate concentration of MT was added. Next, six to eight droplets (30 μl/drop) were made in a dish with a diameter of 60 mm and covered with an appropriate amount of tissue culture oil. Finally, the dish was placed at 37°C with 6% CO 2 and saturated humidity for 18 h of equilibration.

ICSI/IVF insemination
Under a microscope, the cumulus-oocyte complex (COC) in follicular uid was picked up and placed into balanced fertilization medium (Cook, Sydney, Australia) at 37°C with 6% CO 2 and saturated humidity for 4-6 h of culture in vitro, followed by ICSI or IVF insemination.The process of ICSI insemination has been described in detail in our previously published literature [12]. Following ICSI, the inseminated oocytes were directly transferred into cleavage culture medium containing MT for embryo culture in vitro. For IVF insemination, the COC, which underwent 4-6 h of culture in vitro, was transferred into fertilization medium containing 5×10 5 /ml grade A and grade B sperm for 6 h of culture at 37°C with 6% CO 2 and saturated humidity. Next, the granule cells around the oocytes were removed and those with the second polar body, which were considered fertilized oocytes, were selected under an inverted microscope. Finally, all fertilized oocytes were transferred into cleavage culture medium with MT for subsequent embryo culture.

Embryo culture
The oocytes undergoing in vitro culture in the balanced cleavage culture droplets (one oocyte/drop) were observed based on their fertilization status 14-16 h after insemination, and then the culture was continued at 37°C, 6% CO 2, and saturated humidity. Two days later, all of the formed cleavage embryos were transferred into balanced blastocyte culture droplets (one to three embryos/drop) for an additional two or three days of blastocyst culture. Finally, the obtained high-quality blastocysts (see Fig. 2) were selected and cryopreserved in -196°C liquid nitrogen using the vitri cation method. In this process, all embryos formed were scored according to the Tomas [13]and Gardner [14]scoring standards.

Embryo transfer and pregnancy determination
Three months later, the cryopreserved blastocyst was warmed. According to the patient's age, one or two warmed blastocysts were transferred into the uterus under B-ultrasound guidance. Two weeks after embryo transfer, serum hCG levels were examined and a biochemical pregnancy was de ned as a positive hCG value (≥25 IU/L). At seven weeks following embryo transfer, the presence of a gestational sac identi ed by an ultrasound scan was referred to as a clinical pregnancy. For the detailed operation process of embryo vitri cation and warming, please refer to our previously published literature [15].
Statistical analysis SPSS software version 23.0 was used to perform statistical analysis. The differences in means between continuous variables [female age, male age, basic follicle-stimulating hormone (FSH) level, basic luteinizing hormone (LH) level, basic E2 level, duration infertility, average number of oocytes retrieved and body mass index (BMI)] were expressed as the mean ± SD. Categorical data on developmental competence (rates of fertilization, cleavage, high-quality cleavage embryo, and high-quality blastocyst) in each group were analysed using the chi-squared test or Fisher's exact test. P-values <0.05 were considered statistically signi cant.

Results
Screening the optimal MT concentration for embryo culture medium A total of 400 human immature oocytes of 198 COH cycles from 198 patients were collected for IVM culture, and 295 IVM-MII oocytes were obtained. ICSI insemination and embryo culture in vitro were performed, and the detailed results are shown in Table 1. There were no signi cant differences in age, FSH, luteinizing hormone (LH), oestradiol (E2), body mass index (BMI), years of infertility, or number of oocytes obtained in each group (see Table 1). Table 2, after adding 10 -9 M MT to the embryo culture medium, the fertilization, cleavage, blastocyst and high-quality blastocyst rates of the 10 -9 M group were signi cantly different those of the 0 M group (p <0.01). The blastocyst and high-quality blastocyst rates of the 10 -9 M group were signi cantly different from those of the 10 -5 M group (p <0.01, p <0.001), and the blastocyst rate of the 10 -9 M group was signi cantly different from that of the 10 -11 M group (p <0.01). The above data revealed that the addition of 10 -9 M MT to the embryo culture could signi cantly improve the fertilization and in vitro development of the fertilized embryo, and 10 -9 M was the optimal concentration of MT.

As shown in
Fertilization and subsequent embryo development of oocytes in the 10 -9 M and 0 M groups Table 3 shows that in the previous cycle without MT (0 M group), a total of 1838 oocytes were collected, of which 1372 MII oocytes were fertilized by IVF/ICSI, and the fertilization rate was 83.6% (1147/1372).
Among the 140 cycles of 140 patients with subsequent added MT (10 -9 M group), a total of 1658 oocytes were collected, including 1288 MII oocytes, and the fertilization rate after insemination was 87.7% (1129/1288). The fertilization, cleavage, high-quality embryo, blastocyst, and high-quality blastocyst rates in the 10 -9 M group were signi cantly different from those in the 0 M group ( Oocyte fertilization and subsequent embryo development in the 10 -9 M and 0 M groups of patients who experienced 1 failed IVF/ICSI cycle As shown in Fig. 3, there were 1116 oocytes in the 10 -9 M group and 1009 oocytes in the 0 M group of patients who experienced a single failed IVF/ICSI cycle. In terms of fertilization rate (90.1% vs. 88.2%) and cleavage rate (94.6% vs. 92.9%), the rates of the 10 -9 M group were higher than those of the 0 M group, but these differences were not signi cant. The high-quality embryo rate (56.8% vs. 39.4%) and high-quality blastocyst rate (42.0% vs. 25.3%) of the 10 -9 M group were signi cantly higher than those of the 0 M group (both p <0.0001).
The result of oocyte fertilization and subsequent embryo development in the 10 -9 M and 0 M groups of patients who experienced 2 failed IVF/ICSI cycles As shown in Fig. 4, there were a total of 468 oocytes in the 10 -9 M group and 688 oocytes in the 0 M group of patients who experienced two failed IVF/ICSI cycles. The fertilization, cleavage, high-quality embryo and high-quality blastocyst rates of the 10 -9 M group were higher than those of the 0 M group.
The result of oocyte fertilization and subsequent embryo development in the 10 -9 M and 0 M groups of patients who experienced ≥3 failed IVF/ICSI cycles As shown in Fig. 5, there were a total of 74 oocytes in the 10 -9 M group and 141 oocytes in the 0 M group of patients who experienced three or more failed IVF/ICSI cycles. The fertilization, cleavage, high-quality embryo and high-quality blastocyst rates of the 10 -9 M group were higher than those of the 0 M group.

Clinical transfer outcomes
By the end of this manuscript submission, there had been 50 cycles of vitri ed-warmed embryo transfers conducted the experimental group. A total of 61 warmed blastocysts were transferred, and the average number of transfers was 1.22. Thirty-two cases achieved biochemical pregnancy, and clinical pregnancy was con rmed in 20 cases. The implantation rate, biochemical pregnancy rate and clinical pregnancy rate were signi cantly higher than those of the 0 M group (65.6% vs. 9.7%, p <0.0001; 64.0% vs. 12.5%, p <0.0001; 40.0% vs. 11.7%, p <0.0001) (see Table 4). Notably, to date, two women delivered two healthy new-borns, including a male and a female, by caesarean section at 38 weeks of gestational age. Apgar scores were 10 and 9; the body weights were 2.7 and 2.5 kg, and the body lengths were 53 and 51 cm, respectively. The physical and mental development of these infants was normal at their regular postnatal follow-up and the other 18 women who achieved clinical pregnancy also had good examination indexes.

Discussion
In the process of ART treatment, patients encounter many cycles of treatment failure due to poor embryo quality. There are many factors affecting embryo development, such as culture medium [16], CO 2 concentration [17], temperature [17], in vitro operations [18,19], and sperm and oocyte quality [20,21]. It can be considered that during ART treatment, the quality of oocytes is one of the most critical factors that determine the embryo status and clinical outcome. Any factor affecting the quality of the oocyte will affect its fertilization and subsequent embryo development [22] and, ultimately, the cycle outcome of ART treatment. In this study, an appropriate concentration of MT was added to the embryo culture medium and the embryo development and clinical treatment outcomes of the patients were observed, to understand whether the application of MT has a positive impact on embryo development.
According to research reports, embryos are in a relatively high oxygen environment during in vitro culture, which will cause oxidative stress due to an imbalances, antioxidant defence system, resulting in a large amount of ROS produced by oocytes and embryos during development, thus affecting the quality of oocytes and outcome of embryo development in vitro [23]. MT is a highly effective antioxidant with strong antioxidant activity. Animal studies have shown that the addition of MT to embryo culture medium can improve embryo development in vitro, and it has been con rmed that the improvement is concentration dependent [4]. In 2020, our team found that adding MT to human IVM medium can improve the IVM outcome of human immature oocytes by promoting mitochondrial function and inhibiting damage due to oxidative stress, and 10 -5 M was found to be the optimal concentration [10].
Based on the conclusions of previous studies, two main questions were posed. (1) Can adding MT to the culture medium of human embryos improve the in vitro development and clinical outcome of embryos? (2) Is its effect also correlated with concentration? Therefore, in this study, immature human oocytes in the COH cycle were collected for IVM to obtain IVM-MII oocytes and ICSI insemination was conducted.
Next, the fertilized oocytes were cultured in vitro in culture medium containing 0, 10 -11 , 10 -9 , 10 -7 or 10 -5 M MT, and a systematic comparative analysis was performed of the fertilization of oocytes and early embryo development in each group was performed. The fertilization, cleavage, blastocyst and highquality blastocyst rates of the 10 -9 M group were all signi cantly higher than those of the 0 M group. The blastocyst and high-quality blastocyst rates of the 10 -9 M group were signi cantly different from those of the 10 -5 M group, and the blastocyst rate was also signi cantly different from that of the 10 -11 M group. In addition, the fertilization, cleavage, blastocyst and high-quality blastocyst rates of the 10 -9 M group were higher than those of the 10 -7 M group, but these differences were not signi cant. It has been reported that the fertilization rate, cleavage rate and total blastocyst cell number of porcine IVF embryos cultured in medium containing 10 -9 M MT were signi cantly increased [24]. Wang et al. [4] added different concentrations of MT to the culture medium of bovine embryos in vitro and found that 10 -9 M MT could signi cantly promote embryo development. These results are consistent with the results of the present study. These results showed that the addition of 10 -9 M MT to human embryo culture medium could signi cantly improve the fertilization of human oocytes and embryo development in vitro obtained after fertilization, and 10 -9 M was the optimal concentration. Next, 10 -9 M was selected as the MT concentration to be added to the embryo culture medium of patients with failed IVF/ICSI cycles for embryo culture in vitro. The study found that in terms of the fertilization, cleavage, high-quality embryo, blastocyst and high-quality blastocyst rates, the values of the 10 -9 M group were signi cantly higher than those of the 0 M group. In this study, further group analysis was also carried out according to the number of failed IVF/ICSI cycles and it was discovered that the application of 10 -9 M MT to embryo culture in vitro could signi cantly improve embryonic development in the repeated cycle of each group. A large number of studies have found that adding MT to the culture medium can reduce the levels of ROS in oocytes and promote embryonic development of cattle, mice, sheep, and pigs [7,[25][26][27]. Nakano et al. [28] con rmed that the addition of MT during embryo culture in vitro can reduce the level of ROS in parthenogenetic embryos and promote embryo development. These ndings are consistent with the results of the current study, suggesting that the improvement of embryonic development in patients with failed IVF/ICSI cycles by MT may be related to the highly effective antioxidant properties of MT. Oxidative stress inhibits oocyte maturation and embryo development, and MT has a strong antioxidant effect, which can resist oxidative stress, maintain the balance of the antioxidant system, reduce ROS content in the embryo, and promote gamete maturation and embryo development. In our previous study, we found that MT can improve the embryo development and clinical outcome of IVM-MII, and successfully delivered healthy new-borns, which proved the feasibility of MT in clinical treatment [9,10]. In this study, to date, 50 cycles of vitri ed-warmed embryo transfers with MT intervention have been performed. The implantation rate, biochemical pregnancy rate, and clinical pregnancy rate of the 10 -9 M group were signi cantly higher than those of the 0 M group, and two healthy new-borns were successfully delivered. This result shows that the application of 10 -9 M MT to embryo culture medium in vitro can signi cantly improve the clinical treatment effect of patients with failed IVF/ICSI cycles and ultimately achieve the birth of healthy offspring, which greatly enhances our con dence in treating such patients and thereby increases the overall success rate of ART treatment.
In conclusion, the application of MT to embryo culture medium in vitro can improve embryonic development for patients with repeated cycles after failed IVF/ICSI cycles and lead to good clinical outcomes. The optimal concentration of MT was 10 -9 M.

Declarations
Ethics approval and consent to participate This study was approved by the Ethics Committee of Anhui Medical University(2015013). Before conducting the study, the partners of all enrolled patients were interviewed and provided signed informed consent.

Consent for publication
All authors provided nal approval of the version to be published and agree to be accountable for all aspects of the work in ensuring that questions.

Availability of data and materials
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Competing interests
The authors declare that there are no con icts of interest.  Values are presented as mean ± standard deviation.    Experimental ow chart. o.n, oocyte number; c.n, cycle number; p.n, patient number.