The exact role of sperm ROS in early embryonic development has yet to be fully identified. Conventional evidence indicates two significant adverse impacts of sperm ROS. First, high levels of ROS directly impair sperm function. ROS are often generated in spermatozoa as a result of normal metabolic activity , and low levels of ROS are required for functions such as mobility, acrosome reaction and fertilization . Spermatozoa are especially vulnerable to oxidative stress because of their limited antioxidative capacity. A series of oxidative stress reactions occur when sperm ROS exceed the upper limit of total antioxidative capacity, and those reactions can damage the sperm mitochondrial membrane, which will further increase ROS production and lead to mitochondrial dysfunction . Second, the oxidative stress microenvironment of oocytes caused by sperm ROS transfer into oocytes during fertilization procedures and sperm DNA damage induced by ROS may have adverse effects on early embryo development, such as embryo growth retardation, stagnation or even apoptosis, eventually affecting clinical outcomes [15, 16, 17]. Much of the existing research agreed that increased ROS level was detrimental to embryo development, no matter the study subjects were human beings or animals.
However, previous clinical studies considered that sperm ROS levels cannot completely predict embryo viability and clinical outcome . Possible reasons could include the following. 1. Different types of sperm ROS may have different effects on embryo development. 2. The calculation methods of embryo evaluation indices exhibit some irrationality. For example, the parameters of fertilization rate and embryo development are calculated with the number of retrieved oocytes as the denominator in standard practice, including immature and abnormal oocytes. The result is that adverse effects caused by oocytes are falsely attributed to sperm ROS, resulting in data deviations. 3. There are several confounding factors affecting embryo development and clinical outcomes, especially female factors . 4. Significant differences in sperm ROS were observed due to the varying degrees of oocyte quality [12, 13].
Therefore, several strategies were adopted in this study. 1. The two indices of sperm mROS and hydrogen peroxide were detected simultaneously to observe the differences between different types of ROS on embryo development. 2. The "effective number of oocytes" was defined as mature oocytes observed after IVF routine fertilization for 17–19 h for calculation. The objective was to reduce the interference of some oocyte-derived factors on embryo development. 3. We used multivariate data analysis (covariates included female baseline demographics, endocrine state, COH protocol and dosage of Gn, etc.) to analyze the influence of sperm ROS on embryo development from linear and nonlinear perspectives. 4. Patients were divided and analyzed according to infertility etiology, and the effects of sperm ROS on embryo development in patients with normo-ovulatory and anovulatory infertility were comprehensively studied.
The three major findings of the current study are as follows: 1. low levels of sperm hydrogen peroxide are conducive to blastocyst formation; 2. Sperm mROS affects mainly cleavage-stage embryo development; 3. The influences of sperm mROS on cleavage-stage embryos show very interesting opposing effects depending on whether the female patient has normal ovulation and an appropriately high level of sperm mROS, promoting embryo development in patients with anovulatory infertility but having negative effects in those with normo-ovulatory infertility. The findings that high levels of sperm mROS promote cleavage-stage embryo development in patients with anovulatory infertility has not been previously reported in the literature. The generation of this kind of phenomenon should be inseparable from oocyte quality in patients with anovulatory infertility.
The cause of ovulatory dysfunction is complex. At present, the literature on the relationship between human ROS and embryo development in female anovulatory patients has focused on PCOS. The points of research were concentrated to observation of phenomena between embryo development and ROS originating from follicular fluid , embryo culture media [20, 21], granulosa cells [22, 23, 24] and sperm .
Many studies have found that the oocyte utilization rate of PCOS patients is low after IVF cycles. Oxidative stress exists in follicular fluid and influences follicular growth and development. Oxidative damage interferes with the normal metabolic function of oocytes and granulosa cells and reduces the support and protection of granulosa cells to oocytes [26, 27]. Inflammation and mitochondrial dysfunction can increase the accumulation of ROS in granulosa cells [22, 23], which could affect oocyte development and fertilization, even embryo implantation . These studies indicate that oocytes in PCOS infertility have been in a state of high activation of oxygen metabolism. However, according to the data of this study on the relationship between sperm mROS and cleavage-stage embryo, we speculate that the oxidative stress state of the follicular fluid microenvironment and granulosa cells may not be equal to the real situation of oxidative stress in oocytes and embryos. However, due to regulatory and ethical concerns, it is difficult to obtain human oocytes for scientific research. Furthermore, there is a lack of direct evidence to test mitochondrial ROS metabolism in oocytes surrounded by granulosa cells in PCOS infertility. The effect on and mechanism of different types of sperm ROS transfer to the oocyte during fertilization and subsequent embryo development remain unclear. In addition, from the perspective of sperm, our research data showed that the correlation between selected sperm mROS and hydrogen peroxide was weak (r = 0.125, P < 0.01). these results indicated that although sperm mROS and hydrogen peroxide are both ROS substances, their influences on embryo development may not be synchronized.
One study showed that mitochondrial dysfunction existed in germinal vesicle (GV) and metaphase II oocytes of a insulin-resistant PCOS mouse, and MII oocytes displayed a decrease in the ATP content and the mitochondrial DNA (mtDNA) copy number and an increase in the hydrogen peroxide level  Another research reported that mitochondrial energy deficiency in mouse oocytes resulted in a decreased oocyte nuclear maturation rate and ROS generation, but did not affect the fertilization rate .
Why does an appropriate amount of high-level sperm mROS promote cleavage-stage embryo development in patients with ovulation disorders? We speculate that this is due to the low level of mitochondrial reactive oxygen species in the mature oocytes of patients with ovulation disorder. Mitochondrial dysfunction may exist in the oocytes of patients with infertility due to ovulation disorders, especially in PCOS patients, and ATP in short supply may lead to insufficient mitochondrial ROS production. Indicated by the animal experiments, sperm mitochondria are distributed among the blastomeres after fertilization, gradually decrease with embryo proliferation, and sperm mitochondria are undetectable at the blastocyst stage [30, 31]. The suitable concentration of mitochondrial ROS is an important signaling messenger and has been shown to promote the proliferation and differentiation of the embryo. Reducing the mitochondrial ROS level of the pig embryo will weaken embryo mitochondrial biogenesis . We hypothesized that mROS carried by sperm are distributed among the blastomeres along with sperm mitochondria after fusing with oocytes, which may just fill the gap of oocyte mROS. However, low level or excess elevation of sperm mROS transferring into the oocyte may result in insufficient or excessive embryo mROS, which is not conducive to cleavage-stage embryo growth. Therefore, the deductions need to be further confirmed.
Previous researchers used the luminol-peroxidase chemiluminescence method to test human sperm hydrogen peroxide and observed that hydrogen peroxide had a negative effect on blastocyst development . Another study found that the hydrogen peroxide level in day 3 culture medium was negatively correlated with IVF/ICSI blastocyst formation . These conclusions are basically consistent with the result of our study. However, the sensitivity of the luminol peroxidase detection system for the detection of extracellular reactive oxygen species in non-leukocyte pollution, being less than that of flow cytometry, is insufficient .
Multiple logistic regression analysis indicated that sperm ROS had a significant effect on early embryo development in the current study, but the OR value was not very high. This is due to the complexity of factors affecting embryo development; on the other hand, the logistic regression analysis model was used only for linear data analysis. We further revealed the complicated effects of sperm ROS on embryo development through multifactor RCS analysis. First, sperm mROS and hydrogen peroxide presented a nonlinearity in embryonic development in patients with ovulation disorders; that is, sperm ROS promoted or inhibited embryo development only at an appropriate level, and sperm ROS at excessively high or low levels were disadvantageous to embryonic growth. The effect of mROS on cleavage-stage embryos was positive, and that of hydrogen peroxide on blastocyst formation was negative (Fig. 1A-C). Second, the effects of sperm ROS showed two mixed modes, both linear and nonlinear, but the linear effect existed mainly in patients with normo-ovulatory infertility. Generally, sperm ROS has a negative effect (Fig. 1D-I); specifically, a high level of sperm ROS is not conducive to embryo growth, which is consistent with most research results . The RCS analysis showed that the cleavage-stage embryo developed much better when the mROS content of male selected sperm was in range of 2.4% – 8.5% in patients with ovulation disorders, overall. A sperm mROS level less than 2.2% was good for embryo growth in patients with normo-ovulatory infertility; the hydrogen peroxide level below 0.4% in selected sperm was beneficial to blastocyst formation regardless of whether the patient had an ovulation disorder.
There are also some limitations in this research. For example, the findings remain to be confirmed further from a clinical point of view. Additionally, the methods used for sperm ROS assays in this study lack quantitative analysis of ROS.