Hydrogen sulfide and its role in female reproduction

Hydrogen sulfide (H2S) is a gaseous signaling molecule produced in the body by three enzymes: cystathionine-β-synthase (CBS), cystathionine-γ-lyase (CSE) and 3-mercaptopyruvate sulfurtransferase (3-MST). H2S is crucial in various physiological processes associated with female mammalian reproduction. These include estrus cycle, oocyte maturation, oocyte aging, ovulation, embryo transport and early embryo development, the development of the placenta and fetal membranes, pregnancy, and the initiation of labor. Despite the confirmed presence of H2S-producing enzymes in all female reproductive tissues, as described in this review, the exact mechanisms of H2S action in these tissues remain in most cases unclear. Therefore, this review aims to summarize the knowledge about the presence and effects of H2S in these tissues and outline possible signaling pathways that mediate these effects. Understanding these pathways may lead to the development of new therapeutic strategies in the field of women’s health and perinatal medicine.


Introduction
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 (H 2 S), was demonstrated (3).H 2 S 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 H 2 S (4,5).
Three enzymes are responsible for the endogenous production of H 2 S, namely cystathionine-β-synthase (CBS), cystathionine-γ-lyase (CSE) and 3-mercaptopyruvate sulfurtransferase .In addition to these, cysteine aminotransferase (CAT) is sometimes mentioned as the fourth H 2 S-producing enzyme (Figure 1) (6,7).The main substrate for the enzymatic production of H 2 S is L-cysteine (8) (Figure 1), although physiologically, H 2 S can also be generated from D-cysteine (9).However, H 2 S 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 H 2 S. H 2 S 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 H 2 S is the reproductive tract.H 2 S 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 H 2 S is the facilitation of erection (12, 13).In the female reproductive system, H 2 S has been detected in oocytes (14), follicular cells at all stages (15), the uterus (16), and the placenta (17).In female reproduction, H 2 S 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 H 2 S also participates (20,21).H 2 S production also occurs in the vagina and clitoral smooth muscle, where it supports smooth muscle relaxation, vaginal lubrication, and epithelial ion transport (22).

Molecular targets of H S
The effects of H 2 S on various molecular targets are summarized in Figure 2. The first confirmed target of H 2 S was cytochrome c oxidase in mitochondria.In high H 2 S concentrations, mitochondrial activity can be inhibited, and thus adenosine triphosphate (ATP) production is prevented.However, in lower concentrations, H 2 S 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 H 2 S-producing enzyme -3-MST (25,26).H 2 S 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 H 2 S intracellular targets during inflammatory processes, as well as during embryonic development.H 2 S 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), H 2 S may have pro-inflammatory effects in NF-κB in/dependent manner (30).Both results point to the influence of H 2 S on inflammatory processes and its tissue specificity.H 2 S 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).

GRAPHICAL ABSTRACT
BioRender was used to create images and diagrams.Anabolic pathways of H 2 S. H 2 S is enzymatically produced in the body by three enzymes: CBS, CSE, and 3-MST, which also requires CAT for H 2 S production.The main substrate for H 2 S formation is L-cysteine, which can, under the influence of H 2 S-producing enzymes, be produced from homocysteine (Hcy) supplied by the folate and methionine cycles.The image illustrates various pathways involved in the endogenous production of H 2 S in the body under the influence of CBS, CSE, and 3-MST, as well as the byproducts of these biochemical reactions.Molecular targets of H 2 S that should be noticed are cellular proteins themselves.An important effect of H 2 S is the S-sulfhydration of proteins.This process involves the delivery of a sulfur atom derived from the H 2 S 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, H 2 S 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, H 2 S also regulates other channels such as largeconductance calcium-activated potassium ion channels (BK Ca ) (52), L-type and T-type Ca 2+ 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 Ca 2+ (60) and BK Ca channels (52,61).Inhibited ion channels via S-sulfhydration are L-type Ca 2+ (45,53,62), T-type Ca 2+ (54) and BK Ca channels (63).

Detection of H 2 S-producing enzymes and the role of H 2 S in female reproductive tissues
Over the last two decades, H 2 S-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 H 2 S-producing enzymes in these tissues.
Among the initial experiments investigating the function of H 2 S in the female reproductive system, knockout studies (CBS-KO; CSE-KO) have been described (64)(65)(66)(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 H 2 S 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 H 2 S-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 H 2 S 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 H 2 S, 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 H 2 S formation.CBS utilizes Hcy or L-cysteine for H 2 S 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 H 2 S 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 H 2 S levels.However, further experiments are necessary to understand CBS's role in female reproduction precisely.

The role of H 2 S in oocytes
The influence of H 2 S on oocyte maturation (19,80,81), ovulation (15,82), and embryo transport to the uterus (83) has been studied in mice and human oocytes, particularly in connection with luteinizing hormone (LH), which increases CSE production in granulosa cells (82).Inhibition of CSE leads to a reduced number of ovulating follicles and corpus luteum and a higher number of unovulated follicles with retained oocytes (64,65,82).LH likely stimulates H 2 S production in granulosa cells in the preovulatory period (82).Furthermore, the regulation of H 2 S through a donor increased the levels of proteins essential for cumulus-oophorus (CO) expansion and follicle rupture (82, 84, 85).These results highlight the connection between the hormonal regulation of female reproduction and H 2 S production (68).
Regarding the role of H 2 S in oocyte maturation, it has been hypothesized that CBS acts as a mediator between the oocyte and granulosa cells and it may contribute to the proper flow of Hcy in follicular cells, and subsequently support the stability of oocyte transmethylation (15,80).H 2 S plays a role during oocyte maturation in the intracellular environment of the oocyte as well.H 2 S regulates signalling pathways during the cell cycle, likely through S-sulfhydration (35).As was mentioned above, H 2 S-mediated regulation has been described in the cAMP-PKA, PI3K-PKB, MAPK and maturation promoting factor (MPF) pathways (43,46,72,84).Using H 2 S donor (Na 2 S), the supporting effect of the H 2 S on oocyte maturation has been proven, as the Na 2 S accelerated the porcine oocyte nuclear maturation and increased MPF activity during GVBD stage.Moreover, this donor increased the number of zygotes with formed pronuclei after the parthenogenetic activation of porcine oocytes (81,84,85).During the germinal vesicle stage (GV), CBS is distributed into the nucleus of oocytes, however, from germinal vesicle breakdown (GVBD) to metaphase II, it is localized around the mitotic spindle, where it is probably essential for acetylation of α-tubulin and proper assembly of the mitotic spindle (Figure 3).Conversely, deletion of the CBS gene leads to meiosis arrest, abnormalities in both the meiotic spindle and chromosome structure and disruption of the kinetochore-microtubule attachment (14).Additionally, H 2 S is produced by cumulus cells, which likely promotes CO expansion (19,86).The importance of H 2 S during oocyte maturation is further supported by the findings of Gelaude et al. (72), who confirmed the effect of H 2 S on meiosis in amphibian oocytes.It has been previously described that H 2 S has anti-aging effects and promotes the longevity, health, and condition of many organ systems, including the fetal membranes, probably through the mammalian target of rapamycin (mTOR) signaling pathway and its downstream factor S6 kinase beta-1 (S6K1) (87)(88)(89).For this reason, a series of experiments describing the role of H 2 S during oocyte aging have been reported.H 2 S-producing enzymes are active in porcine oocytes, and there is a statistically significant decrease in endogenous H 2 S production during the first day of aging.Inhibition of H 2 Sproducing enzymes induces signs of aging in oocytes and significantly increases the number of fragmented oocytes (Figure 3) (90).Conversely, an exogenous H 2 S donor (Na 2 S) can reverse these manifestations.Cultivation in the presence of the H 2 S donor can also positively affect subsequent embryonic development after parthenogenetic activation (90).These results were supported by research confirming reduced CBS expression in oocytes and ovaries of old mice (14).The mechanism of H 2 S action on oocytes involves the regulation of K ATP and L-type Ca 2+ channels, which play a crucial role during oocyte aging through S-sulfhydration.H 2 S activates K ATP channels, delaying cell death, and conversely inhibits L-type Ca 2+ channels, which have the opposite effect on oocytes (45,53,62,91).In conclusion, H 2 S is crucial in most processes occurring in oocytes (Figure 3) and their immediate environment.

Uterine vessels
Given the vasodilatory effects of H 2 S (58, 92, 93), this function has been investigated concerning the regulation of blood flow in uterine vessels, which affects the exchange of nutrients and respiratory gases between the mother and the fetus, consequently influencing fetal growth and health (94,95).It appears that the activity of H 2 Sproducing enzymes and the subsequent effect of uterine blood vessel vasodilation are essential, as elevated levels of Hcy (and thus a probable deficiency in H 2 S-producing enzymes) lead to uterine artery blood flow resistance (96).Vasodilatory effects of H 2 S have been confirmed in human (97), sheep (98), and rat (99) uterine arteries, as well as in human umbilical arteries and veins, with this effect occurring primarily during the proliferative phase of the menstrual (estrus) cycle and in gravidity (Figure 4) (20).The mechanism of vasodilation in the vascular system generally occurs through K ATP channels (62, 100, 101).The same mechanism is employed in uterine vessels, as was confirmed by subsequent studies describing an increased number of K ATP channels in human and sheep smooth muscle cells of uterine arteries during pregnancy (20, 102).However, Li et al. (103) contributed to this topic by elucidating the regulation of BK Ca channels by H 2 S in human uterine arteries, so it is conceivable that multiple types of ion channels contribute to the vasodilation of uterine arteries by H 2 S.
In the past decade, studies have emerged reporting the regulation of uterine vessel vasodilation by estrogens through their influence on promoting H 2 S synthesis via CBS and CSE (104).For example, it has been described that during estrogen-dominant phases of the female cycle (i.e., proliferation, pregnancy), CBS production is higher than the secretory phase.Specifically, CBS seems to be the primary H 2 S-producing enzyme responding to elevated estrogen levels, as the expression of CSE and 3-MST does not change in gravid tissue compared to non-gravid tissue (20, 105).Interestingly, Zeigler et al. ( 106) found a decrease in plasma H 2 S levels in the later stages of pregnancy compared to postpartum.This could be explained more likely as an increase in H 2 S consumption, as it is essential for S-sulfhydration of proteins necessary for the growth of maternal and fetal tissues.S-sulfhydrated proteins are extensively involved in processes such as the contraction and relaxation of smooth muscle in blood vessels (107,108).Additionally, during pregnancy, the H 2 S dilution is more significant as the volume of maternal blood plasma can increase by up to 50% (106,109).This hypothesis is supported by the increased production of H 2 S in intrauterine tissues during pregnancy, which leads to a higher rate of S-sulfhydration of proteins compared to non-pregnant tissue.These results support the finding that the expression of CBS is greater in estrogen-dominant phases, as the consumption of H 2 S is also higher (110).
While the precise mechanism describing estrogen-induced stimulation of H 2 S biosynthesis in uterine arteries is unknown, a hypothesis suggests estrogen receptors' important role in this signaling pathway (104).This hypothesis has been recently confirmed, as it was found that estrogen receptors activate CBS promoters, thereby stimulating its production.In contrast, the activity of the CSE promoter remains unchanged (110).When it comes to vascular dilation, it is worth noting the previously established influence of another gasotransmitter -NO, which is also a potent vasodilator in the bloodstream and interacts with H 2 S in many organ systems (111)(112)(113).It is presumable that H 2 S, NO, and estrogens, which interact with both H 2 S and NO, synergistically contribute to the vasodilation of uterine vessels and that these systems behave towards each other as backup mechanisms because pathology occurs only after the inhibition of both signaling pathways (111, 113).

Uterus and pregnancy
One of the most referred impacts of H 2 S on the human (114) and rat (115) uterus is its tocolytic effects, which can be caused by H 2 S itself, as well as its precursor (L-cysteine) (116) or donor (NaHS) (117).These effects, promoting uterine relaxation, are significant for gravidity maintenance.Therefore, it is not surprising that the expression of CBS and CSE and production of H 2 S increases during gravidity and, conversely, abruptly decreases with the onset of labor (110,114,118).H 2 S also effectively prolongs the duration of labor and reduces the frequency of uterine contractions, which can contribute to a smooth delivery process (Figure 4) (119).It is assumed that the mechanism of the tocolytic effects of H 2 S lies in the opening of channels, as the body utilizes the exact mechanism in the bloodstream and other smooth muscle tissues (18,120).Additionally, it has been demonstrated that the inhibition of K ATP channels leads to the absence of relaxation effects of H 2 S donors (45,92).
It is possible that H 2 S also regulates activity of BK Ca channels and L-type Ca 2+ channels, as they also influence the relaxation of myometrium (52,73,118).Furthermore, the tocolytic effects of H 2 S may lie in inhibition of contraction-associated proteins (CAPs) and suppressing the toll-like receptor 4 (TLR4)/NF-κB signaling pathway (Figure 5) (16, 29,42).Besides its tocolytic effects, H 2 S may also impact uterine immune response and placental vessel remodeling through the modulation of the uterine natural killer (uNK) cells (121-123).H 2 S signaling is also essential for maintaining early pregnancy, and its deficiency can lead to reduced litter size due to early embryo loss or placental inflammation (70, 124).H2S may further facilitate the physiological implantation of the embryo by regulating ion transport activity in the endometrial epithelium and supporting DNA synthesis (125)(126)(127).
The relationship between H 2 S and estradiol (E2) is intriguing because both contribute to uterine quiescence during pregnancy by regulating the expression of CAPs (128,129).Estrogens, in general, appear to regulate H 2 S-producing enzymes, consequently affecting the levels of H 2 S itself (98).The increased production of CBS and H 2 S in the uterine arteries during pregnancy is influenced by endogenous estrogens acting through specific estrogen receptors (ER) in pregnant rats.This indicates that the physiological changes associated with pregnancy, such as elevated levels of endogenous estrogens, play a role in stimulating the expression of CBS and subsequent H 2 S production in the uterine arteries.The specific ER-mediated mechanism implies that ER are involved in regulating this process, highlighting the importance of endogenous estrogen signaling in mediating vascular adaptations during pregnancy (110).Specifically, E2 modulates gene expression and redox balance in the uterus by inducing transsulfuration via CBA and CSE, for which this Comparison of H 2 S levels and their functions during pregnancy and labor.In estrogen-dominant phases, such as pregnancy or the proliferative phase of the estrus/menstrual cycle, there is an increased production of H 2 S. In uterine tissues, H 2 S promotes dilation of uterine blood vessels, contributing to tissue perfusion and proper development of the placenta, fetus, and nutrient supply to the fetus.It also maintains the integrity of fetal membranes and has tocolytic effects on the uterus, thereby delaying labor.Before and during labor, there is a significant reduction in H 2 S in uterine tissues, leading to constriction of uterine blood vessels, rupture of fetal membranes, and increased uterine contractions.metabolic pathway is unique (20, 130).The effects of E2 may also influence the metabolism of myometrial cysteine, which is utilized by H 2 S-producing enzymes to generate H 2 S, particularly during periods of elevated E2 levels such as estrus and gravidity.This pathway is mediated through sulfur amino acids and myometrial cysteine sulfinic acid decarboxylase (CSAD), the activity of which is reduced by E2.Estrogen-mediated regulation of H 2 S-producing enzymes and H 2 S itself occurs not only in uterine tissue but also in uterine vessels, where E2 activates CBS promotors leading to increased production of H 2 S in estrogen-dominant phases, leaving no doubt about the connection between H 2 S and estrogens (20, 105).H 2 S-producing enzymes plays a vital role in intrauterine tissues by regulating Hcy levels and thus preventing pathological conditions.Uncontrolled Hcy levels can lead to hyperhomocysteinemia (73, 77) associated with various adverse outcomes in pregnancy, including impaired implantation (131), reduced litter size (124,131), neural tube defects (132), miscarriages (5,64), preeclampsia (5,133,134), hypertension (76,134), and fetal growth restrictions (135).However, CSE is not a secondary enzyme in this matter.Is it also capable of generating H 2 S from Hcy and effectively regulating its levels (Figure 1) (74, 76,77,136,137).Interestingly, CBS-KO in the uterus itself is not a direct cause of infertility in these individuals.Infertility in CBS-KO individuals occurs due to the resulting hyperhomocysteinemia or due to the action of another factor in the uterine environment of CBS-KO homozygotes.This indicates that the prominent role of H 2 S-producing enzymes during pregnancy is regulating Hcy levels around the growing fetus (64).It is worth noting that although CBS-KO may lead to reduced fertility or even infertility in female offspring, this is not the case for male offspring (64,122,131).It is possible that the effect is related to the pathways of female sex hormones, such as LH and E2, as described in previous sections, and may not necessarily affect male fertility.However, further research would be needed to confirm these assumptions.
Given that H 2 S-producing enzymes play a specific role during pregnancy, it can be assumed that the gas they produce also plays a role.In the context of previously mentioned pathogenic states, H 2 S likely inhibits the soluble fms-like tyrosine kinase-1 (sFlt1), a vascular endothelial growth factor (VEGF) antagonist associated with hypertension and preeclampsia (138).Because elevated Hcy levels are a risk factor for preeclampsia, H 2 S may also prevent the onset of preeclampsia through this pathway (18, 73, 133, 139).

The role of H2S in placenta
Like the uterus, a reduced CBS expression towards the end of gestation has been described in placental and decidual tissues.H 2 S likely serves to maintain the integrity of the chorion/amnion before birth by slowing down the aging of the fetal membranes' cells, so it is not surprising that its expression in these tissues decreases with the onset of labor (Figure 4) (18,89).It is also interesting to note that both CBS and CSE expression in fetal membranes decreases much more during physiological labor than in infants delivered by cesarean section (17,140).This shows that H 2 S is necessary for maintaining pregnancy, and a decrease in its expression appears to be one of the critical factors leading to the physiological onset of labor (16, 140).However, it should be noted that the role of H 2 S in the placenta may vary between species.For example, hypoxic conditions in the human placenta lead to increased H 2 S production, which is not observed in rat placenta (18).H 2 , S also contributes to proper placental development by promoting angiogenesis through placental growth factor (PIGF), VEGF, and signaling pathways PKB, nitric oxide synthases (NOS)/NO, and MAPK3/1 (141-143).VEGF is a key factor in regulating placental angiogenesis and this process is stimulated by activation of MAPK pathway in placental endothelial cells (144).However, in contrast to these positive effects of H 2 S, an association has been described between increased CBS expression in placentas and infants with Down syndrome, indicating that proper regulation of H 2 S expression in intrauterine tissues is crucial for physiologically ongoing gravidity (145)(146)(147).
The relationship between H 2 S and two other gasotransmitters in fetal membranes is intriguing.While the CO donor (hemin) in fetal membranes does not affect H 2 S production, the NO donor (sodium nitroprusside) leads to a significant increase in H 2 S production in this tissue (18).It is, therefore, possible that both H 2 S and NO synergistically contribute to maintaining the integrity of fetal membranes and pregnancy.This would imply that intrauterine tissues can be included among many other tissues where a mutual relationship between H 2 S and NO has been observed (112,(148)(149)(150)(151). Mechanisms leading to the tocolytic effect of H 2 S. The tocolytic effects of H 2 S can be mediated by the opening of ion channels (K ATP , BK Ca , L-type Ca 2+ ), as well as through the cGMP pathway or a reduction in intracellular pH.H 2 S achieves these effects by inhibiting CAPs, pro-inflammatory cytokines, and the TLR4/NF-κB signaling pathway.For several years, it has been known that the human trophoblast produces H 2 S through the expression of CBS and CSE, with some studies indicating that CSE is the primary H 2 S-producing enzyme in the first trimester of pregnancy (33, 143).Generally, supplementing the culture medium with H 2 S and NO donors supports embryo development in vitro.Once again, the synergy between these two gasotransmitters was described in this tissue, as H 2 S 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 H 2 S 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 H 2 S, 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 H 2 S modulates genes encoding proteins involved in early embryo epigenetic regulation (152).Even though the precise mechanism of embryonic epigenetic regulation by H 2 S is unknown, it can be assumed that H 2 S 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 H 2 S-producing enzymes may lead, for example, to reduced PIGF production causing fetal growth restriction (FGR) or recurrent spontaneous miscarriages (124,155).
Furthermore, H 2 S 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.H 2 S likely acts against contractile endothelins, facilitating oviduct peristalsis and, consequently, the transit of the embryo itself (83).H 2 S 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 H 2 S expression is also essential in preventing the development of intrauterine growth restriction (IUGR) and preeclampsia (155).Additionally, H 2 S protects the heart of chicken embryos by regulating myocardial K ATP channels (156).
H 2 S-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 H 2 S is essential in embryonic development across various taxa.

Conclusion
The production of H 2 S 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 H 2 S 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 H 2 S and its capability to preserve the integrity of fetal membranes.
Additionally, H 2 S 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 H 2 S-producing enzymes, CBS and CSE, is their ability to regulate homocysteine levels in the vicinity of cells through the production of H 2 S.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 H 2 S signaling may be associated with various pathological conditions.It has been reported that aberrant H 2 S 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/H 2 S signaling significantly contributes to increased embryonic resorption in mice (124).Notably, H 2 S production was found to be upregulated in the human oviduct in ectopic pregnancy, suggesting the involvement of dysregulation of H 2 S homeostasis (83).Dysregulation of H 2 S signaling has been also linked to the pathogenesis of preeclampsia (5,140).Abnormal H 2 S signaling has recently been reported to be involved in diabetes-related uterine dysfunction as it was found that in non-obese diabetic mice, uterine H 2 S production is 2-fold higher than in the control group.This increase in H 2 S 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 H 2 S 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 Ca 2+ , K ATP , BK Ca ), transcription factors (NF-κB), and other cellular messengers (NO, E2, PIGF, cytokines).A particularly interesting function of H 2 S 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 H 2 S 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 H 2 S 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.H 2 S 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.

Funding
The author(s) declare financial support was received for the research, authorship, and/or publication of this article.This review was supported by the Internal Grant Agency of the Czech University of Life Sciences in Prague (SV22-10-21230).

FIGURE 2
FIGURE 2 Molecular targets of H 2 S.These targets can be divided into the following groups: (a) Influence of H 2 S on energy metabolism and cytochrome c oxidase activity; (b) activation and inactivation of various types of ion channels, likely through S-sulfhydration; (c) influence on cell signaling through transcription factors and kinases; (d) modification of a wide range of proteins through S-sulfhydration of cysteine thiol sites; (e) reduction of oxidative stress in mitochondria.ATP, adenosine triphosphate; SSH, S-sulfhydration; NF-κB, nuclear factor-kappa-B; Nrf-2, nuclear factor E2-related factor 2; STAT3, signal transducer and activator of transcription 3; MAPK, mitogen-activated protein kinase; PKA, protein kinase A; PKB, protein kinase B; PKC, protein kinase C; PI3K, phosphoinositide 3-kinase; PDE, phosphodiesterase; LDH, lactate dehydrogenase; GSH, glutathione; GSSG, glutathione disulfide.

FIGURE 3
FIGURE 3Effects of H 2 S on oocytes.H 2 S produced in the ovaries and surrounding reproductive tissues has the following effects: (a) H 2 S promotes ovulation of a more significant number of follicles and the development of a greater number of corpora lutea compared to inhibiting its production (b); (c) H 2 S supports CO expansion; (d) H 2 S promotes oocyte maturation through the regulation of oocyte signaling pathways.Additionally, its production regulates Hcy levels, supports oocyte transmethylation, and ensures proper spindle assembly; (e) H 2 S promotes follicle rupture and, therefore, oocyte ovulation itself; (f) H 2 S delays oocyte aging and reduces the number of fragmented oocytes; its inhibition, on the other hand, leads to an increase in the number of fragmented oocytes; (g) H 2 S supports embryo transport to the uterus; (h) LH stimulates H 2 S production in granulosa cells of follicles, likely through CSE, contributing to the processes mentioned above.

TABLE 1
Detection of H 2 S-producing enzymes in female reproductive tissues.