Several decades ago, hydrogen sulfide was considered only as a toxic gas. However, after the discovery of endogenous production of nitric oxide (NO) (1) and carbon monoxide (CO) (2) in the organism and their effects on various tissues, a third endogenously produced gasotransmitter, hydrogen sulfide (H₂S), was demonstrated (3). H₂S is now known to be involved in a wide range of physiological processes, including reducing cellular oxidative stress, regulating the cell cycle and apoptosis, participating in inflammatory processes, and vasodilating blood vessels (4). The regulation of the nervous and reproductive systems are among the other described functions of H₂S (4, 5).

Three enzymes are responsible for the endogenous production of H₂S, namely cystathionine-β-synthase (CBS), cystathionine-γ-lyase (CSE) and 3-mercaptopyruvate sulfurtransferase (3-MST). In addition to these, cysteine aminotransferase (CAT) is sometimes mentioned as the fourth H₂S-producing enzyme (Figure 1) (6, 7). The main substrate for the enzymatic production of H₂S is L-cysteine (8) (Figure 1), although physiologically, H₂S can also be generated from D-cysteine (9). However, H₂S can also be produced through non-enzymatic processes, such as its production by microorganisms in the digestive tract that metabolize sulfur or the simple dissociation of sodium hydrosulfide (NaHS) into H₂S. H₂S can also be released from acid-labile sulfur, which serves as a reservoir of this molecule in the body (10, 11).

One of the many organ systems affected by H₂S is the reproductive tract. H₂S has been detected in both male and female reproductive tracts of mammals, fish, and amphibians. In the male reproductive tract, one of the most fundamental roles of H₂S is the facilitation of erection (12, 13). In the female reproductive system, H₂S has been detected in oocytes (14), follicular cells at all stages (15), the uterus (16), and the placenta (17). In female reproduction, H₂S is essential during gravidity and labor initiation (17, 18), in oocyte maturation and ovulation (19). It also influences the vasodilation of uterine and placental vessels, thereby affecting the nutrition of the growing embryo/fetus, in whose epigenetic regulation H₂S also participates (20, 21). H₂S production also occurs in the vagina and clitoral smooth muscle, where it supports smooth muscle relaxation, vaginal lubrication, and epithelial ion transport (22).

The effects of H₂S on various molecular targets are summarized in Figure 2.The first confirmed target of H₂S was cytochrome c oxidase in mitochondria. In high H₂S concentrations, mitochondrial activity can be inhibited, and thus adenosine triphosphate (ATP) production is prevented. However, in lower concentrations, H₂S can supply electrons to the mitochondrial respiratory chain through sulfide quinone oxidoreductase and cytochrome c oxidase (23, 24). In mitochondria, there has been detected the H₂S-producing enzyme – 3-MST (25, 26). H₂S is associated with cellular oxidative stress, as it interacts with glutathione, leading to an elevation in its concentration and the subsequent suppression of oxidative stress in mitochondria (27, 28).

Transcription factors are other H₂S intracellular targets during inflammatory processes, as well as during embryonic development. H₂S donors such as NaHS, S-diclofenac, or diallyl sulfide can inhibit nuclear factor kappa B (NF-κB) activation, thereby suppressing the production of pro-inflammatory cytokines (29). Conversely, under certain conditions (dose, exposure time), H₂S may have pro-inflammatory effects in NF-κB in/dependent manner (30). Both results point to the influence of H₂S on inflammatory processes and its tissue specificity. H₂S likely impacts other transcription-mediated processes, such as proliferation (31) or angiogenesis (32), and it appears to play a crucial role in the epigenetic regulation of genes in early embryos (33).

A variety of kinases are also cellular targets of H₂S. Examples are mitogen-activated protein kinases (MAPK), which H₂S can both activate (19, 34) through S-sulfhydration (35) and inhibit (36, 37). H₂S also activates protein kinase A (PKA) (38, 39), phosphoinositide 3-kinase (PI3K)/protein kinase B (PKB) (40–42) or protein kinase C (PKC) (41). Additionally, to targeting kinases, H₂S also inhibits phosphodiesterase, and consequently regulates the levels of cyclic guanosine monophosphate (cGMP) (43, 44).

Molecular targets of H₂S that should be noticed are cellular proteins themselves. An important effect of H₂S is the S-sulfhydration of proteins. This process involves the delivery of a sulfur atom derived from the H₂S molecule to the thiol group of cysteine residues, leading to the formation of a hydropersulfide group (-SSH) (45, 46). These -SSH cysteines are more reactive than cysteines containing only a thiol group, and S-sulfhydration modifies these proteins (46). Interestingly, in cells, S-sulfhydration is considered a common post-translational modification. Among S-sulfhydrated proteins belong ATP synthase, lactate dehydrogenase, ion channels, phosphodiesterase, and many others (23, 44, 45, 47). In ion channels, H₂S is capable of opening ATP-sensitive potassium channels (K_ATP) in the smooth muscle of arteries (48), myocytes (49), and smooth muscle of the intestine (50) or eye (51). However, H₂S also regulates other channels such as large-conductance calcium-activated potassium ion channels (BK_Ca) (52), L-type and T-type Ca²⁺ channels (53, 54), Cl⁻ channels (55), and transient receptor potential vanilloid and ankyrin channels (TRPV and TRPA) (56, 57). S-sulfhydration can activate some channels while inhibiting others. Among activated channels belong K_ATP (58, 59), Cl⁻ (55), TRPV/TRPA (56, 57), T-type Ca²⁺ (60) and BK_Ca channels (52, 61). Inhibited ion channels via S-sulfhydration are L-type Ca²⁺ (45, 53, 62), T-type Ca²⁺ (54) and BK_Ca channels (63).

Over the last two decades, H₂S-producing enzymes have been detected in various female reproductive tract tissues, spanning different animal models, including humans. Table 1 provide summary of the experiments conducted on this topic across diverse animal species and describe the potential significance of H₂S-producing enzymes in these tissues.

Among the initial experiments investigating the function of H₂S in the female reproductive system, knockout studies (CBS-KO; CSE-KO) have been described (64–67). These studies demonstrate the importance of CBS in the maintenance of placental and uterine weight in females, as well as its indispensability in the maturation of growing follicles (64, 65) (Figure 3). Furthermore, the effect of CBS on the regularity and length of the estrus cycle was proven, which subsequently affects the fertility rate in females (64, 68). However, the absence of CBS does not cause morphological abnormalities on ovulated oocytes or the ovaries themselves (64). Interestingly, after transplanting CBS-KO ovaries into healthy recipients, the fertility of the females was not affected, indicating that the H₂S production through CBS in other reproductive tissues is sufficient but probably not essential for maintaining female fertility (64). As for CSE, the absence of this H₂S-producing enzyme in mice appears to have significantly less effect on the incidence of fertility-related defects, as CSE-KO females were fertile, and their pregnancies progressed normally (65, 67, 69). Recent research focused on the fertility of CSE-KO mice showed that CSE-KO leads to a reduced number of successful pregnancies and a higher pro-inflammatory status of fetuses. This suggests that CBS is not the sole key enzyme in H₂S production in the context of female reproduction (70).

The reason why CBS seems more important for female reproduction in most studies (64, 65, 67) when the final product of both enzymes is H₂S, has yet to be investigated. Potential reasons may include variances in homocysteine (Hcy) and cysteine metabolic pathways or differences in the substrate essential for the H₂S formation. CBS utilizes Hcy or L-cysteine for H₂S production (71, 72), with L-cysteine also generated from Hcy by both CBS and CSE (Figure 1). In reproductive tissues, the prevalence of Hcy may favor CBS (18, 73). CSE primarily uses cystathionine/L-cysteine/cystine as a substrate, but it can also utilize Hcy (74–77). However, the direct production of H2S from Hcy by CSE suggests a potential advantage in following the CBS route, interrupting the reaction at the intermediate product, cystathionine, to regulate both Hcy and H2S levels in the body (Figure 1). This proposition is supported by the fact that hyperhomocysteinemia is a critical factor during pregnancy leading, for example, to preeclampsia, miscarriages, uterine artery blood flow resistance or congenital malformations (73, 76, 78). Furthermore, higher H₂S levels can lead to the inhibition of cytochrome c oxidase in mitochondria (24, 79). It is possible that CBS was evolutionarily favored because it can effectively regulate both Hcy levels in tissues and the H₂S levels. However, further experiments are necessary to understand CBS’s role in female reproduction precisely.

For several years, it has been known that the human trophoblast produces H₂S through the expression of CBS and CSE, with some studies indicating that CSE is the primary

H₂S-producing enzyme in the first trimester of pregnancy (33, 143). Generally, supplementing the culture medium with H₂S and NO donors supports embryo development in vitro. Once again, the synergy between these two gasotransmitters was described in this tissue, as H₂S produced by the trophoblast, like VEGF, stimulates endothelial nitric oxide synthase (eNOS) activation (143). However, the precise role of these gasotransmitters in embryogenesis remains unclear. One of the main roles of H₂S during embryo development is likely epigenetic regulation of embryogenesis, cell cycle, support of DNA formation, and proliferation (152, 153). Based on previous research confirming the regulation of specific promoters by H₂S, for example, in vascular smooth muscle cells (154), it can be hypothesized that this regulation is also functional in mammalian embryo cells. This hypothesis is supported by research confirming that H₂S modulates genes encoding proteins involved in early embryo epigenetic regulation (152). Even though the precise mechanism of embryonic epigenetic regulation by H₂S is unknown, it can be assumed that H₂S has a positive effect on early embryonic development, and it may even be essential for enhancing transcription and modification of specific embryonic genes related primarily to metabolism (33). Conversely, reduced expression of H₂S-producing enzymes may lead, for example, to reduced PIGF production causing fetal growth restriction (FGR) or recurrent spontaneous miscarriages (124, 155).

Furthermore, H₂S appears to be an important factor in transporting the morula from the oviduct to the uterus, as inhibition of CBS expression leads to embryo retention or prolongs its transport. H₂S likely acts against contractile endothelins, facilitating oviduct peristalsis and, consequently, the transit of the embryo itself (83). H₂S also promotes proliferation, migration, cytoskeleton remodeling, and invasion of trophoblast cells, where it activates various types of kinases (e.g., FAK, Src, ERK), Rho GTPases, and upregulates metalloproteinases 2 and 9 (89). On the other hand, excessive expression of CBS and CSE in the oviducts may be a sign of ectopic gravidity or embryonal carcinoma, so it cannot be conclusively stated that higher levels of CBS and CSE expression in this tissue indicate physiological embryo transport (83). Proper regulation of H₂S expression is also essential in preventing the development of intrauterine growth restriction (IUGR) and preeclampsia (155). Additionally, H₂S protects the heart of chicken embryos by regulating myocardial K_ATP channels (156).

H₂S-producing enzymes have been identified even in zebrafish embryos, where there were described 2 cbs orthologs – cbsa and cbsb (157). Cbsb is crucial for ion homeostasis, while cbsa appears redundant (158, 159). These results indicate that H₂S is essential in embryonic development across various taxa.

The production of H₂S has been demonstrated in all female reproductive tissues, primarily through the enzymes CBS and CSE and, to a lesser extent, through 3-MST (Table 1). We can assume that H₂S plays a crucial role in various physiological processes associated with female reproduction, given its ability to vasodilate uterine and umbilical vessels, as well as maintain pregnancy through both the tocolytic effects of H₂S and its capability to preserve the integrity of fetal membranes. Additionally, H₂S has anti-aging effects on mammalian oocytes, supporting their maturation and ovulation, aiding in the transport of early embryos into the uterus, and epigenetic regulation of their genes. Further on, an important characteristic of H₂S-producing enzymes, CBS and CSE, is their ability to regulate homocysteine levels in the vicinity of cells through the production of H₂S. This mechanism within the female reproductive tract serves to prevent pathological conditions such as hyperhomocysteinemia, which can lead to preeclampsia, miscarriages, congenital fetal abnormalities, and others.

Conversely, dysregulation of H₂S signaling may be associated with various pathological conditions. It has been reported that aberrant H₂S metabolism results in impaired oviductal transport of embryos and developmental delay of preimplantation embryos in mice (83). It has also been shown that dysregulated placental CBS/H₂S signaling significantly contributes to increased embryonic resorption in mice (124). Notably, H₂S production was found to be upregulated in the human oviduct in ectopic pregnancy, suggesting the involvement of dysregulation of H₂S homeostasis (83). Dysregulation of H₂S signaling has been also linked to the pathogenesis of preeclampsia (5, 140). Abnormal H₂S signaling has recently been reported to be involved in diabetes-related uterine dysfunction as it was found that in non-obese diabetic mice, uterine H₂S production is 2-fold higher than in the control group. This increase in H₂S associated with 3-MST has been shown to cause a reduction in spontaneous endogenous uterine contractions (160). In addition, CBS has been proposed to promote ovarian cancer progression, tumor growth, and drug resistance (161), while CSE has been associated with breast cancer metastasis promotion (162).

The effects of H₂S and subsequent signaling pathways in the aforementioned tissues are well-described. These effects are mediated by kinases (PKA, PKB, MAPKs), ion channels (T and L-type Ca²⁺, K_ATP, BK_Ca), transcription factors (NF-κB), and other cellular messengers (NO, E2, PIGF, cytokines). A particularly interesting function of H₂S is its epigenetic effects, involving chromatin modification and activation of specific promoters, as well as its interaction with female sex hormones (LH, E2). Yet, these effects are not sufficiently elucidated, although clarifying their precise molecular aspects might result in the development of new methods and drugs, particularly in the field of women’s health and perinatal medicine.

In conclusion, a comprehensive understanding of H₂S function could lead to its therapeutic use in disorders related to reproduction. For instance, its tocolytic and vasodilatory effects could be utilized to maintain pregnancy, support embryo implantation, and prevent miscarriages. The interaction of H₂S with LH and E2 could also be used in the development of new drugs regulating the menstrual cycle or supporting superovulation in women undergoing oocyte aspiration prior to in vitro fertilization. H₂S could also enhance the culture media of oocytes and embryos in IVF clinics, promoting their proper development and increasing the chances of successful pregnancy. Additionally, it may serve as an effective treatment for conditions like hyperhomocysteinemia, preeclampsia, or irregular estrus/menstrual cycles.

AP: Conceptualization, Writing – original draft, Writing – review & editing. ZP: Conceptualization, Writing – original draft, Writing – review & editing. BK: Writing – review & editing. NZ: Writing – review & editing. EC: Writing – review & editing. PP: Supervision, Writing – review & editing. MS: Supervision, Writing – review & editing.