Therapeutic Potential of Hydrogen Sulfide in Reproductive System Disorders

Abstract

Hydrogen sulfide (H₂S), previously regarded as a toxic exhaust and atmospheric pollutant, has emerged as the third gaseous signaling molecule following nitric oxide (NO) and carbon monoxide (CO). Recent research has revealed significant biological effects of H₂S in a variety of systems, such as the nervous, cardiovascular, and digestive systems. Additionally, H₂S has been found to impact reproductive system function and may have therapeutic implications for reproductive disorders. This paper explores the relationship between H₂S and male reproductive disorders, specifically erectile dysfunction, prostate cancer, male infertility, and testicular damage. Additionally, it examines the impact of H₂S regulation on the pathophysiology of the female reproductive system, including improvements in preterm birth, endometriosis, pre-eclampsia, fetal growth restriction, unexplained recurrent spontaneous abortion, placental oxidative damage, embryo implantation, recovery of myometrium post-delivery, and ovulation. The study delves into the regulatory functions of H₂S within the reproductive systems of both genders, including its impact on the NO/cGMP pathway, the activation of K⁺ channels, and the relaxation mechanism of the spongy smooth muscle through the ROCK pathway, aiming to broaden the scope of potential therapeutic strategies for treating reproductive system disorders in clinical settings.

1. Introduction

The reproductive system, as one of the most important components of the human body, is mainly divided into the male and female reproductive systems and is an important basis for human survival and reproduction. The organs of the reproductive system are categorized according to their location, including the penis, testicles, prostate gland, vas deferens, and epididymis in males [1], whereas women are mainly composed of the fallopian tubes, ovaries, uterus, and vagina [2]. The structurally intact and normal function of the reproductive system of both sexes is essential for the health and reproduction of both sexes. Environmental contaminants, microorganisms, and poor lifestyles can lead to structural damage or dysfunction of the reproductive system [3,4,5,6,7,8]. In addition, it is also associated with various reproductive disorders such as decreased fertility, erectile dysfunction (ED), varicocele, preterm labor, pre-eclampsia, and fetal growth restriction. Due to the complexity and diversity of the pathogenesis of diseases of the reproductive system, there are no clear and effective measures to stop the progression of the diseases. Therefore, there is an urgent need to address this issue and to identify possible diagnostic and therapeutic targets for related diseases.

Hydrogen sulfide (H₂S) is the third endogenous gaseous signal molecule discovered after nitric oxide (NO) and carbon monoxide (CO). It is involved in the regulation of multiple organ systems, including the reproductive system [9,10], and biological processes such as angiogenesis, inflammation, oxidative stress, autophagy, and apoptosis [11,12,13]. H₂S generation systems have been found in the mammal reproductive systems [14,15,16]. With regard to its role in the male reproductive system, H₂S promotes the relaxation of human cavernous smooth muscle, mediates erectile function [17,18,19,20], enhances spermatogonial proliferation [21,22], and regulates the relaxation of vas deferens smooth muscle [23,24]. Among the effects of H₂S on the female reproductive system, H₂S suppresses the natural contraction of uterine bands taken from pregnant rats [25], notably relaxes vaginal smooth muscle in rabbits [16], modulates oviductal transport [26], and causes placental metamorphosis [27]. Altogether, H₂S is significant in the reproductive systems of both men and women and could potentially be beneficial in treating reproductive system disorders. This review provides a comprehensive summary of the distribution, role, and mechanisms of H₂S in the male and female reproductive systems, with the aim of providing a theoretical basis for the discovery of new therapeutic targets and the development of more effective therapeutic strategies for reproductive system diseases.

2. Physical and Chemical Properties of H₂S

H₂S is a small-molecule gasotransmitter that is highly lipophilic and can, therefore, penetrate cell membranes without the aid of specific transporter proteins or receptors. In the human body, H₂S exists mainly as a hydrosulfide ion (HS-) and, to a lesser extent, as a free gas. Through a sequence of reactions, it is possible for it to undergo oxidation and produce sulfur dioxide, sulfates, and elemental sulfur, as well as various other substances [28,29]. A dynamic equilibrium is maintained between H₂S and HS to ensure the stability of H₂S in vivo and maintain the normal pH of the internal environment. In mammals, endogenous H₂S is produced through desulfurization of cysteine, a process that involves three major tissue-specific enzymes, namely, cystathionine-β-synthase (CBS), cystathionine gamma-lyase (CSE), and 3-mercaptopyruvate sulfurtransferase (3-MPST) [30,31]. Both CBS and CSE are localized in the cytoplasm, whereas 3-MPST is mainly localized in mitochondria [32,33]. Endogenous H₂S is generated from L-cysteine (L-Cys) and homocysteine by CBS and CSE [34,35]. In addition, it can be generated from 3-mercaptopyruvate by 3-MPST. 3-mercaptopyruvate is produced from either L-Cys (CAT/MPST pathway) by cysteine aminotransferase (CAT) or D-cysteine (DAO/MPST pathway) by amino acid oxidase (DAO) [36,37]. H₂S is metabolized through the following three mechanisms in vivo: (1) Mitochondrial oxidation: H₂S is first catalyzed by sulfide quinone oxidoreductase (SQR) at the inner mitochondrial membrane to form glutathione persulfide (GSSH), which is then oxidized to sulfite by persulfide dioxygenase (ETHE1) in the mitochondrial matrix. Sulfite can be further oxidized to sulfate by sulfite oxidase or reacted with GSSH to form thiosulfate under the transsulfuration of thiosulfate:cyanide sulfurtransferase (TST, rhodanese) [38,39]; (2) Cytoplasmic methylation: H₂S is metabolized in the cytoplasm by thiol-S-methyltransferase (TMST) to methyl mercaptan, which is subsequently converted to dimethyl sulfide [40,41]; (3) Plasma molecule binding: Free H₂S in plasma can bind to glutathione disulfide (GSSG) or other metal- or disulfide-containing molecules, such as hemoglobin, to form sulfate or H₂S conjugates such as sulfhemoglobin (see Figure 1) [42,43]. The metabolite excretion occurs primarily via the kidney, spleen, and lungs. In the body, H₂S levels remain controversial. The estimated physiological levels of circulating H₂S range from 10 to 100 μM [44], whereas the concentration of peripheral H₂S is estimated to be 30–100 μM [45], which may be lower in the reproductive system, with only nanomolar levels in the corpus cavernosum [46].

3. Distribution of H₂S Synthases in the Reproductive System

3.1. Distribution of H₂S Synthases in the Male Reproductive System

H₂S generated within the male reproductive system was first identified in rabbit cavernous smooth muscle [47]. Shortly thereafter, d’Emmanuele et al. [17] demonstrated that CBS and CSE were found to produce endogenous H₂S in the human corpus cavernosum (HCC) and identified the location of the two enzymes via immunohistochemical analysis of tissues. CSE was found in peripheral nerves and vascular smooth muscle cells (SMCs) of the penile and trabecular muscular tissue of HCC, while CBS was primarily found in trabecular muscular tissue. Both CBS and CSE were determined in the human prostate, with CBS substantially localized in the lumen and epithelial cells and CSE in the periacinar stroma [48,49]. The species and tissue distribution of H₂S synthases expressed in other mammals, such as rats and mice, differ slightly from those in humans (Figure 2). In rat penile tissue, cavernous smooth muscle cells (CCSMCs) express CSE, CBS, CAT, DAO, and 3-MPST but lack the expression of CBS [50,51]. CBS, CSE, and 3-MPST are predominantly expressed in the mouse corpus cavernosum, especially in luminal endothelial cells and vascular endothelial cells. However, immunohistochemical findings have shown that the expression of CSE is higher than that of CBS and 3-MPST [52]. In addition, only CBS is expressed in the luminal subcutaneous smooth muscle, vascular smooth muscle, and peripheral nerves of the corpus cavernosum [53]. In a study, five enzymes that produce H₂S were found in the prostates of rats. CBS, MPST, and CAT were mainly localized in glandular epithelial cells. In contrast to MPST expression, CBS expression was significantly higher in PR-V than in PR-D, whereas CAT expression was not significantly different between the two sites [36]. CSE is essential for H₂S synthesis in the mouse prostate, with its expression decreasing with age [54].

To date, most studies investigating the distribution of H₂S in the testis have used animal models. In rats, CBS is mainly discovered in the interstitial cells of seminiferous tubules. And it is moderately expressed in immature germ cells in the marginal region of seminiferous tubules but rarely expressed in mature germ cells. CSE is abundant in the vascular wall of the testicular interstitium, supporting cells, and immature germ cells [21]. In mice, three H₂S-generating enzymes are expressed in testicular germ cells [55]. In humans, mice, and rats, CSE and CBS are mainly localized in the luminal epithelium and muscular tissue of the vas deferens [23]. In rats, CBS and CSE mRNA levels are higher in the somatic and caudal regions of the epididymis, with CBS identified in epithelial cells and CSE in SMCs below the epithelium [56].

3.2. Distribution of H₂S Synthases in the Female Reproductive System

Patel et al. [57] were the first to examine the production of endogenous H₂S and the distribution of CBS and CSE in human and rat uterine tissues. They qualitatively detected the expression of CBS and CES in the non-pregnant uterus, pregnant uterus, placenta, and fetal membranes of rats and the chorionic villi, amnion, and myometrium of the human placenta using protein blotting. Subsequent studies have indicated that 3-MPST is present in the human placenta, with CBS and CSE predominantly found in syncytiotrophoblasts and vascular endothelial cells in the chorionic villi. Meanwhile, 3-MPST was observed in syncytiotrophoblasts [58,59]. In addition, CSE is expressed in trophoblasts and mesenchymal cells within the chorionic core of human placental villi and in the SMCs of chorionic trunk arteries [60,61]. Unlike the expression of CBS and CSE, the expression of 3-MPST in placental tissues does not show a significant difference between a healthy female and a female with pre-eclampsia [59]. A similar expression of CBS and CSE is observed in the endothelial cells and SMCs of human uterine arteries [62,63]. In addition, 3-MPST and CAT are expressed in human uterine arteries. The levels of CBS mRNA and protein are increased during gestation and the proliferating stage of the menstrual cycle; however, CSE, 3-MPST, and CAT protein expression do not undergo significant changes during these periods [62]. In human endometrium, the expression of CBS is upregulated during these two periods, whereas that of CSE is not significantly altered. CBS and CSE are primarily found in the epithelium, stroma, and microvessels within the human endometrium. However, CBS is specifically located in stromal cells and blood vessels during pregnancy and the proliferative stage of the menstrual cycle [64]. In mouse ovaries, CBS is expressed in follicular cells at all stages, with its expression being higher in primary and primordial follicles than in secondary and sinus follicles. After follicular development, granulosa cells are divided into cumulus cells and mural granulosa cells, and high levels of CBS are observed in both subpopulations. However, CBS is not found in oocytes [15]. In human fallopian tubes, CBS and CSE are determined in the tubal epithelium [26]. Similarly, both enzymes are expressed in human vaginal tissues. Sun et al. [65] found that CBS and CSE are predominantly discovered in rat vaginal epithelial cells (VK2/E6E7), and CSE was expressed in rat vaginal vessels by immunohistochemical analysis (Figure 3).

4. Effects of H₂S on Male Reproductive System Disorders

4.1. H₂S Promotes Penile Erection

4.1.1. H₂S Relieves Erectile Dysfunction

Penile erection relies mainly on smooth muscle relaxation in the corpus cavernosum and penile arteries for its morphology and function. Upon stimulation, parasympathetic nerves release neurotransmitters that relax the smooth muscles in the corpus cavernosum and penile arteries. In response to this relaxation, blood flows into the cavernous sinus, causing it to swell and compress the veins, thereby reducing blood outflow and raising intracavernous pressure (ICP), which ultimately results in an erection [66]. Several studies have demonstrated that H₂S promotes penile erection. Jupiter et al. [67] reported that the H₂S donors Na₂S and NaHS promoted penile erection and prolonged the duration of erection in anesthetized rats in a concentration-dependent manner. According to Srilatha et al. [18], injecting NaHS into non-human primates’ cavernous bodies notably elevated penile length and ICP. These changes were consistent with those observed before penile erection was induced by the injection of prostaglandin E1. Another study showed that injection of the CSE inhibitor DL-propargylglycine (PAG) into the external jugular vein of rats inhibited H₂S production, consequently decreasing ICP induced by the electrical stimulation of cavernous nerves. It was also shown that injecting rats with PAG, an inhibitor of CSE, to suppress H₂S generation reduced electrical stimulation and cavernous nerve-induced ICP. This finding suggests that cavernous nerve excitation mediates penile erection by facilitating the release of H₂S. Consistently, d’Emmanuele et al. [17] demonstrated the presence of a CSE/H₂S-generating system in the peripheral nerves of the human penis.

The erection of the penis is regulated by nerves, blood vessels, and smooth muscles. An injury to one or more parts of the penis can result in erectile dysfunction (ED). It has been determined that rats with ED due to radical prostatectomy, hyperlipidemia, diabetes, or hypertension have reduced levels of H₂S-generating enzyme in their penile tissues [19,51,68,69]. Furthermore, La Fuente et al. reported that ED is associated with defects in the L-cysteine/H₂S pathway [70]. However, supplementation with exogenous H₂S donors can alleviate ED. ACS6 was the first drug, developed from Sildenafil and ADT-OH, able to supplement H₂S [20]. While ACS6 is similar to sildenafil in terms of relaxing the corpus cavernosum at the same dose, its potency in suppressing superoxide formation and PDE5 expression is higher than that of sildenafil citrate or NaHS. Notably, treatment with ACS6 for a period could ameliorate ED through suppressing oxidative stress and decreasing PDE5 levels. There is evidence that certain extractions from plants stimulate H₂S generation in vivo. For instance, resveratrol (RVT) [71] induces the relaxation of corpus cavernosum (CC) in mice in a concentration-dependent manner. Inhibitors of CBS counteract RVT’s effects, but not those of eNOS. In response to RVT, the production of H₂S is increased at both basal and L-cysteine-induced levels. It appears that RVT-stimulated CC relaxation is partly dependent on H₂S production and is not dependent on NO production. Furthermore, it was discovered that sodium tanshinone IIA sulfonate (STS) [69] reversed the downregulation of CBS and CSE and inhibited H₂S production in rats fed a high-fat diet (HFD). Additionally, STS guarded against ED through the activation of the Nrf2/HO-1 pathway in response to HFD-induced oxidative stress.

In regards to treating ED, preferred medications such as sildenafil and tadalafil (also known as PDE-5is) are available [72]; however, their efficacy is poor in some cases. β3 adrenergic receptors are found in the SMCs of HCC. Upon activation, these receptors result in smooth muscle relaxation in HCC under the influence of cGMP [73]. An activator of the β3 adrenergic receptor, BRL37344, was discovered to relax HCC and the penile artery, whereas PAG reversed these relaxation effects [74]. Treatment of both HCC bands and tissue homogenates from penile arterial rings with BRL37344 significantly increased H₂S production; however, inhibition of H₂S generation prevented the BRL37344-mediated increment of cGMP level in HCC and penile arterial tissues. These results suggest that activation of β3 adrenoceptors leads to relaxation of HCC and penile arteries in an H₂S/cGMP-dependent manner. Given that selective agonists of β3 adrenoceptors, such as mirabegron [75], function independently of the endothelium, they can be used as replacement medications for these individuals who have no effect on PDE-5 therapy.

Because H₂S is mainly released by SMCs and is independent of endothelial function, it represents a potential treatment option for ED related to endothelial malfunction, such as ED related to diabetes and metabolic syndrome (metS). High-glucose diet-induced metS has been shown to reduce H₂S production in rat penile tissues [76]. However, exogenous administration of H₂S donors can improve erectile function. A study showed that GYY4137, a slow-releasing H₂S donor, significantly improved the CC vascular responsiveness by restraining the TGF-β1/Smad/CTGF signal pathway in STZ-mediated diabetic rats [77]. Furthermore, H₂S donors combined with PDE-5is hold potential applications for treating ED. Combining NaHS and tadalafil therapy was more effective than monotherapy against ED in some rat models of bladder outlet obstruction [78]. The reduced erectile response and decreased H₂S levels were only partially restored after monotherapy but were completely restored after combination therapy. In addition, combination therapy counteracted changes in the morphology and function of the penis triggered by bladder outlet blockage-induced ischemia and improved the erectile response. These results suggest that H₂S donors can alleviate ED and restore the spontaneous erectile response with long-term use.

4.1.2. Mechanisms of H₂S in Regulating Penile Erection

Priapism is caused and maintained by more arterial blood inflow than venous return. Vascular smooth muscle relaxation plays a key role in priapism. Although the exact mechanism through which H₂S relaxes CC smooth muscle remains unclear, it has been reported to involve the synergetic action of NO, stimulation of K⁺ channels, as well as the modulation of RhoA/ROCK (

Table 1. Effects of H₂S on the reproductive system.

Organ	Action	Mechanisms	Models	References
Penis	Promotion of erection	Activation of RhoA/ROCK pathway and KATP channel	HCC strips from transsexual procedures (n = 6)	[17]
		Activation of RhoA/ROCK pathway	Bilateral cavernous nerve injury rats (n = 18)	[19]
		Dependent on cAMP or cGMP	Rabbit CC strips (n = 5)	[47]
		Activation of BKCa channel	Anesthetized rat	[67]
		Increase eNOS expression	L-NAME-induced hypertensive rats (n = 40)	[68]
		Activation of sGC/cGMP pathway	HCC strips of men with ED (n = 50)	[81]
		Increase the levels of NO and HO activity	Diabetic rats (n = 90)	[82]
		Activation of NO/sGC/cGMP pathway	CSE−/− mice	[83]
		Activation of BKCa channel and Kv channel	Rat CC strips	[84]
		Activation of NO/sGC/cGMP pathway and KATP channel	STZ-diabetic rats (n = 10 or 12)	[85]
		Activation of RhoA/ROCK pathway	Mouse CC strips	[86]
	Improve vascular injury of CC	Inhibition of TGF-β1/Smad/CTGF pathway	STZ-diabetic rats	[77]
Prostate	Inhibition of CRPC	S-sulfhydrylation of AR	CSE knock-out and overexpression LNCaP; LNCaP-B	[54]
	Promotion of NE differentiation	Enhance the activity of Cav3.2	LNCaP	[87]
Testis	Alleviate the apoptosis of testicular germ cells	Increase SOD activity and reduce Bax/Bcl-2 ratio	Heat exposure treated mice	[55]
	Reduce sperm motility	Activation of AMPK/Akt related pathways	Boar sperm;NH4Cl and/or Na2S treated mice	[88]
		Activation of Keap1/Nrf2 signaling pathway	GC-2spd(ts) cells derived from mouse spermatocytes	[89]
		Reduction of iNOS, TNF-α, and Apaf-1	Testicular torsion-induced I/R injury rat model (n = 38)	[90]
	Alleviate testosterone synthesis	Sulfhydrylation of PDE4A/8A and activation of cAMP/PKA pathway	LPS+ induced testosterone synthesis impairment model of mouse Leydig tumour cells	[91]
	Increase sperm motility	Activation of CBS/H2S pathway	LPS− and diabetes-induced sperm dysfunction mice model;CBS+/− mice model	[92]
		Scavenge ROS	Fe2+/ascorbate treated boar sperm	[93]
Epididymis	Maintain quiescence of epididymal sperm	Activation of KATP channel and BKCa channel	Cauda epididymal epithelium cells	[56]
	Alleviate varicocele-induced epididymis injury	Activation of PI3K/Akt pathway	Experimental varicocele rat model (n = 60)	[94]
Vas deferens	Regulation of VD spontaneous contraction	Activation of L-Cys/H2S pathway	Human VDs from monorchidism surgery (n = 3); rat VDs (n = 20); mice VDs (n = 11)	[23]
		Activation of BKCa channel	Rat VDs	[24]
Uterus	Abnormal contraction of uterus	Activation of L-Cys/CSE/H2S	L-Cys and sildenafil treated uterus collected from CSE−/− mice (n = 10).	[74]
		Activation of KATP channel	L-Cys treated Human myometrium (n = 6).	[95]
	Premature delivery	Inhibition of TLR4/NF-κB signaling pathway	LPS-induced C57BL/6 mice (n = 8).	[96]
			IL-1β treated USMCs obtained from TL and TNL myometrium (n = 12).
		Inhibition of ERK1/2 and NF-κB signaling pathway	LPS-induced BALB/c mice (n = 10).	[97]
	Promote uterine angiogenesis	Improve the expression of CBS	UA collected from female uterus (NP, n = 20; P, n = 10);	[62]
			Phenylephrine-treated UA collected from SD mice (n = 8–10).
			E2-treated HESCs isolated from human endometrial	[64]
			(POM, n = 9; pPRM, n = 13; sPRM, n = 4; Preg, n = 13).
			ERT-treated UA isolated from ovx NP ewes (n = 3–5).	[98]
			E2-treated UASMCs isolated from late pregnant ewes (n = 3–5).	[99]
	Endometriosis	Activation of NF-κB	HESCs isolated from human endometrium (n = 21); Allogeneic endometrial fragment transplantation induced BABL/C mice (n = 6).	[100]
	Decreased uterine fertility	Replenish the CBS	Ovary transplantation treated CBS−/− mice.	[101]
Placenta	Preeclampsia	Activation of CSE/H2S pathway	Placenta and plasma collected from women with PE and normotensive pregnant (n = 14); PAG-treated C57BL/6 pregnant mice.	[60]
		Activation of KATP channel	IUGR and PE placenta collected from female (n = 6 and 13).	[61]
		Promotion of NO synthesis	Hypoxia-treated placental explants; bicarbonate buffer treated healthy placenta (n = 8).
		Activation of L-cys/H2S pathway	Placenta collected from women with PE (n = 10); L-Cys treated SHR.	[102]
		Inhibition of SFlt-1 expression	Placenta obtained from woman with PE (n = 19);	[59]
			NaHS and L-Cys treated syncytiotrophoblasts.
			Placenta collected from women with PE (n = 18);	[103]
			NaHS and L-Cys treated cytotrophoblasts
		Inhibition of TLR4 expression	Plasma collected from women with PE (n = 30);	[104]
			DOCA-salt and LPS treated SD pregnant rats (n = 6).
		Activation of VEGF expression	Placenta obtained from women with PE (n = 19); NaHS and L-Cys treated explants and trophoblasts isolated from healthy placentas.	[105]
		Activation of PI3K/Akt1/eNOS and ERK1/2 signaling pathway	NaHS-treated oFPAECs isolated from sheep placental arteries.	[106]
			BCA and CHH treated HTR-8/SVneo.
	Promotion of embryo implantation	Inhibition of CFTR expression	AOAA or PAG treated pregnant KM rat (n = 3);	[107]
			NaHS-treated MEECs isolated from KM female rat.
	Intrauterine fetal growth restriction	Activation of CSE/H2S pathway	HR-induced SVA collected from human IUGR placentas (n = 34).	[108]
	Recurrent spontaneous abortion	Activation of CBS/H2S pathway	AOAA and PAG treated C57BL/6 mice; GYY4137 and NaHS treated CBS+/−♀ mice;	[109]
			NaHS or L-Cys treated HTR8/SVneo and JEG3 cells.
	Promotion placental development	Elevation of Nrf2 expression	Cigarette smoke induced SD pregnancy rats.	[110]
	Gestational diabetes	Inhibition of NLPR3	Placenta collected from pregnant women with GDM (n = 16);	[111]
			NaHS or L-Cys treated HTR-8/SVneo.
Ovary	Promotion of ovulation	Elevation of CBS and CSE expression	PMSG-treated granulosa cells isolated from ICR mice ovary.	[112]
Fallopian tube	Promotion of tubal transport	Activation of H2S signaling pathway	NaHS or L-Cys treated fallopian tubes collected from female (n = 7 or 5);	[26]
			AOAA-treated C57BL/6J mice and BALB/c mice (n = 22).
Vagina	Sexual response	Activation of CAMP, NO-cGMP, KATP signaling pathway	NaHS-treated Vaginal isolated from white rabbits (n = 12).	[16]
		Activation of CSE/H2S pathway	NaHS-treated SD rats (n = 10); NaHS-treated VK2/E6E7.	[65]

Abbreviation: L-Cys: L-cysteine, LPS: lipopolysaccharide, USMCs: uterine smooth muscle cells, TL: term in labour, TNL: term no labour, HESCs: human endometrial stromal cells, POM: postmenopausal, pPRM: proliferative premenopausal, sPRM: secretory premenopausal, Preg: pregnant, ERT: estrogen replacement therapy, UA: uterine artery, OVX: ovariectomized, NP: nonpregnant, P: pregnant, E₂: estradiol-17β, UASMC: uterine artery smooth muscle cell, SD: Sprague Dawley, PE: preeclampsia, SHR: sponta hypertensive rats, PAG: DL-propargylglycine, HUVECs: human umbilical vein endothelial cells, IUGR: intrauterine fetal growth restriction, oFPAECs: ovine fetoplacental artery endothelial cells, BCA: β-cyano-L-alanine, CHH: carboxymethyl hydroxylamine hemihydrochloride, DOCA: desoxycorticosterone acetate, AOAA: amino-oxyacetic acid, KM: kunming, MEECs: murine primary uterine endometrial epithelial cells, HR: hypoxia reoxygenation, SVA: stem villus arterie, WT; wild-type, GDMz; gestational diabetes mellitus, PMSGzzz; pregnant mare’s serum gonadotropin, Akt: protein kinase B, AMPK: adenosine 5′-monophosphate AMP-activated protein kinase, Apaf-1: apoptosis protease activating factor-1, CRPC: castration-resistant prostate cancer, BKCa: large-conductance Ca²⁺-activated K⁺, cAMP: cyclic adenosine monophosphate, cGMP: cyclic guanosine monophosphate, CTGF: connective tissue growth factor, CC: corpus cavernosum, ED: erectile dysfunction, eNOS: endothelial NOS, ERK1/2: extracellular signal-regulated kinase 1/2, HCC: human corpus cavernosum, HO: heme oxygenase, iNOS: inducible NOS, Keap1: Kelch-like ECH-associated protein 1, L-NAME: nω-nitro-L-arginine, NE: neuroendocrine, NF-κB: nuclear factor-κB, PCa: prostate cancer, ROS: oxygen species, Nrf2: nuclear factor erythroid 2-related factor 2, PI3K: phosphatidylinositol 3′-OH kinase, ROCK: Rho-kinase, sGC: soluble guanylate cyclase, SOD: superoxide dismutase, STZ: streptozotocin, TGF-β1: transforming growth factor-β1, TNF-α: tumor necrosis factor-α, VD: vas deferens.

), with NO playing the most controvertible role. Penile erection is thought to be mediated primarily by NO [79]. It promotes the production of cGMP, an intracellular second messenger, by activating soluble guanylate cyclase (sGC), which in turn relaxes corpus cavernosum smooth muscle by regulating calcium channels and intracellular contractile proteins [80]. Several studies have shown that H₂S can increase the expression of NOS, the key enzyme for NO production. Meng et al. [81] showed that eNOS activity, protein and mRNA expression, and NO levels were higher in rat cavernosum tissues treated with NaHS (1 mM) than in control tissues. Yilmaz et al. [68] found that administration of NaHS (0.037mg/kg) counteracted the decrease in eNOS and nNOS protein levels in penile tissues. Mostafa et al. [82] found that NaHS (30 mg/kg) increased the levels of NO in the cavernous tissues of rats, which is consistent with the aforementioned findings. However, knockout of CSE significantly reduced the levels of p-eNOS and NO in rat corpus cavernosum tissues [83] (Figure 4). These findings suggest that H₂S regulates penile erectile function by promoting the production of NO.

Some in vivo studies have shown that NO, stimulation of K⁺ channels, and modulation of the RhoA/ROCK pathway do not have synergistic effects. Srilatha et al. [47] pre-contracted isolated rabbit CC bands with norepinephrine and L-NAME, an inhibitor of the NO-generating enzymes, followed by NaHS treatment. The relaxation caused by NaHS was not affected by L-NAME. In another study, cavernous smooth muscle was precontracted in the presence of atropine and then exposed to the CBS inhibitor aminooxyacetic acid (AOAA) or the CSE inhibitor β-cyanoalanine (BCA) or PAG, followed by electrical stimulation. The results showed that inhibition of H₂S synthesis did not affect the NO-mediated relaxation of cavernous smooth muscle. Similarly, treatment with L-NAME did not cause a notable reduction in the relaxation of human corpus cavernosum smooth muscle induced by NaHS (1 μM–10 mM) [17]. The intracavernosal administration of Na₂S (0.03–1 mg/kg) significantly increased ICP, whereas the intracavernosal administration of L-NAME did not significantly affect it [67]. Furthermore, intracavernosal administration of Na₂S did not affect the erectile response caused by SNP. Mice lacking NO exhibit a higher level of CSE and 3-MPST, thereby increasing the production of H₂S and its relaxation effects on the CC [52]. These changes may be compensatory but suggest that the relaxation effects of H₂S are independent of NO. Therefore, H₂S may not synergize with NO to promote the erectile response.

These conflicting results indicate that the mechanism by which the relaxation effects of H₂S on CC smooth muscles in vivo may not be dependent on the NO-cGMP pathway. However, endogenous H₂S inhibits phosphodiesterase (PDE) to suppress cGMP degradation [113]. Several studies have shown that H₂S in the penis regulates cGMP levels by acting on sGC. For example, treatment with L-Cys (1 μM–1 mM) and NaHS (1 μM–1 mM) increases cGMP levels in HCC, whereas administration with ODQ, an inhibitor of sGC, reverses this effect [114]. The decrease in H₂S content in CSE-KO mice weakens the redox state of sGC, thereby downregulating cGMP in the penis [83], suggesting that H₂S promotes penile erection by upregulating cGMP through other pathways.

Potassium channels have been reported to participate in the H₂S-mediated relaxation of corpus cavernosum smooth muscle. There are four basic types of K⁺ channels exhibited in arterial smooth muscle: KCa, K_ATP, Kir, and Kv channels [115]. These four channels are also found in human corpus cavernosum tissues [116]. To examine the role of different K⁺ channels in the H₂S-mediated relaxation of corpus cavernosum smooth muscle, Jupiter et al. [67] injected tetraethylammonium chloride (TEA, a non-selective inhibitor of K⁺ channels), iberiotoxin (an inhibitor of BKCa channels), and glyburide (GLB, an inhibitor of K_ATP channels) into the cavernous bodies of anesthetized rats. These three compounds affected the changes in ICP induced by Na₂S (0.03–1 mg/kg). Intracavernous administration of Na₂S increased ICP, which was counteracted by TEA and iberiotoxin administration; however, the effects of GLB on ICP were not evident. In another study, rat CC bands were treated with TEA, GLB, 4-AP, a Kv channel inhibitor, or BaCl2, a Kir channel inhibitor, in an organ bath, and the effects of these compounds on the CC relaxation caused by L-cysteine were also examined. According to the results, TEA and 4-AP notably weakened the L-cysteine-stimulated CC relaxation effect, with TEA being more effective than 4-AP. However, GLB and BaCl2 did not have significant inhibitory effects on L-cysteine-induced relaxation [84]. The above results indicated that BKCa and Kv channels are associated with H₂S-induced CC relaxation in the rat. GYY4137 relaxes rat CC partially via KATP channels [85]. Another study showed that GLB remarkably attenuated the NaHS-induced relaxation of HCC bands [17], while, as for the concentration of GLB used in the experiment, it is more than 10 times and may have interfered with the results since GLB at more than 10 times suppresses Na⁺-K⁺ pumps and L-type Ca²⁺ channels [117]. Correspondingly, the researchers found that administration of GLB at 10 μM did not inhibit NaHS-mediated relaxation of CC strips in rats [50,84] or humans [114].

ROCK, a serine/threonine kinase that promotes smooth muscle contraction, is involved in the H₂S-mediated relaxation of cavernous smooth muscle. Chitaley et al. [118] found that treatment with Y-27632, an inhibitor of ROCK, increased ICP and stimulated penile erection in rats. These effects were found to be independent of the NO pathway. It was found that NaHS had significantly greater relaxation effects on HCC strips precontracted with the ROCK modulator U46619 or endothelin-1 than phenylephrine on HCC strips [17], implying that H₂S may regulate the ROCK pathway. A ROCK inhibitor, Fasudil, can also inhibit the relaxation effects of H₂S donors on mouse CC, indicating that H₂S may interact with ROCK [119]. Aydinoglu et al. [86] reported for the first time that ROCK was involved in the H₂S-induced relaxation of the corpus cavernosum of mice contracted with phenylephrine. Pretreatment with Y-27632 significantly attenuated the contractile effects induced by phenylephrine-phosphorylating MYPT1 at Thr696 in isolated mouse CC strips. In addition, treatment with L-cysteine or NaHS substantially eliminated the contractile response of phenylephrine. Combining Y-27632 with L-cysteine or NaHS decreased MYPT1 phosphorylation, while PAG or AOAA reversed the inhibition. Additionally, Y-27632 notably elevated H₂S generation at basic levels and in response to L-cysteine, whereas PAG and AOAA inhibited H₂S synthesis, indicating that ROCK may at least partially inhibit CSE and CBS levels in CC tissues [86]. The RhoA/ROCK pathway can promote CC smooth muscle contraction by modulating the CCSMCs phenotype [19]. Researchers have concluded that in rats with bilateral cavernous nerve damage, treatment with NaHS suppressed the phenotypic conversion of CCSMCs caused by the enhancement of the RhoA/ROCK pathway, contributing to the improvement of erectile dysfunction [19].

4.2. Regulatory Role of H₂S in Prostate Cancer

The effects of H₂S on the prostate are mainly examined in the context of prostate cancer (PCa), especially castration-resistant prostate cancer (CRPC). The growth and development of the prostate rely on androgens. At present, one of the most widely used hormone therapies involves the direct blockade of the androgen receptor (AR). H₂S was found to inhibit the trans-activation of the second zinc finger of AR through S-sulfhydration of cysteine at residues 611 and 614 and effectively suppress the growth of PCa cells resistant to anti-androgen therapy [54]. Bicalutamide is an anticancer agent that competitively binds to AR. It was found that CSE expression was substantially lower in bicalutamide-resistant PCa cells (LNCaP-B) than in androgen-dependent PCa cells (LNCaP). In addition, overexpression of CSE or exogenous administration of H₂S (30 μM NaHS) restored the sensitivity of LNcap-B cells to bicalutamide, whereas LNCaP cells with knockdown of CSE continued to grow even in the presence of bicalutamide [54]. These results suggest that the CSE/H₂S system can be used as a valuable prognostic indicator and an effective therapeutic target for early-stage PCa and CRPC. However, some studies have reported that H₂S plays a negative regulatory role in PCa. It was shown that upregulation of cytoplasmic cAMP caused by androgen deprivation increased the generation of the CSE/H₂S system [87]. Subsequently, H₂S promoted androgen-independent proliferation by enhancing the activity of Cav3.2. Both endogenous and exogenous H₂S can promote the function of T-type Ca²⁺ channels in LNCaP cells with neuroendocrine differentiation potential, consequently promoting the progression to neuroendocrine CRPC.

The above results suggest that the CSE/H₂S system may be involved in the regulation of PCa progression. However, the opposite regulatory results may be because the response of cancer cells to H₂S varies with the type of H₂S donor, concentration of the donor, and type of cancer [120]. H₂S-related drugs have been reported to exert good therapeutic effects against PCa. For example, H₂S-releasing non-steroidal anti-inflammatory drugs (HS-NSAIDs), in which traditional NSAIDs are covalently attached to an H₂S-releasing moiety, are 28–3000 times more effective than traditional NSAIDs in inhibiting tumor cell growth in multiple cancers, including PCa [121]. In the castration- and Adriamycin-resistant PCa cell line DU-145, the accumulation of H₂S-releasing doxorubicin (H₂SDox) is significantly higher than that of traditional doxorubicin. Mechanistically, the release of the SH2 group from H₂SDox damages the activity of PCa cells by forming disulfide bonds on P-gp, consequently overcoming Adriamycin resistance [122]. Recently, a tumor microenvironment (TME)-responsive Zn²⁺-interference and H₂S-mediated gas therapy based on tannic acid-modified zinc sulfide nanoparticles (ZnS@TA) was discovered. In a neutral environment (pH = 7.4), the same concentration of ZnS@TA had no significant effect on the viability of DU-145 cells but significantly inhibited their migratory and invasive abilities and enhanced their apoptosis in TME [123].

4.3. H₂S Promotes Male Fertility

When combined with NH3, H₂S in exhaust emissions can disrupt spermatogenesis [124] and reduce sperm motility through AMPK/AKT-related pathways [88], eventually impairing male fertility. However, many studies have shown that H₂S protects the testis and sperm owing to its anti-inflammatory, antioxidative, and anti-apoptotic properties (Figure 5). Heat stress significantly increases the production of ROS and inhibits the activity of superoxide dismutase (SOD) in mouse germ cells. However, treatment with NaHS promotes SOD activity, reduces ROS production, inhibits the release of cytochrome C, and reduces the Bax-to-Bcl-2 ratio, thereby suppressing the apoptosis of testicular germ cells induced by heat exposure [55]. In addition, restraint stress can increase the levels of ROS and malondialdehyde (MDA) and decrease the levels of CBS, CSE, and 3-MPST in rat serum, suggesting that downregulated H₂S plays a key role in male infertility [125].

H₂S has been shown to alleviate testicular dysfunction and germ cell apoptosis caused by toxic substances. A study found that treatment with the antineoplastic drug cisplatin significantly increased MDA levels and decreased SOD activity in rat testicular tissues. However, administration of NaHS counteracted all biochemical, histological, and morphological changes induced by cisplatin [126]. The researchers found that nanoplastics induced ROS-dependent mitochondrial apoptosis and autophagy in GC-2spd(ts) cells derived from mouse spermatocytes, resulting in reproductive toxicity. H₂S alleviated nanoplastic-induced reproductive toxicity by increasing the expression of antioxidant enzymes such as HO-1 and NQO1 through Keap1/Nrf2 signaling [89]. Testosterone deficiency can affect the normal morphological features and motility of sperm and impair male fertility [127]. It was shown that overexpression of CBS restored testosterone levels by promoting the S-sulfhydration of PDE4A and PDE8A and activating the cAMP/PKA pathway in mouse stromal tumor cell (MLTC-1) models of lipopolysaccharide (LPS)- and hydrogen peroxide (H₂O₂)-induced testosterone synthesis impairment [91].

Some studies have shown that sperm motility is decreased in animal models of defective H₂S synthesis; however, exogenous H₂S administration or CBS overexpression increases sperm motility in these models. Impaired spermatogenesis caused by stress is associated with decreased production of endogenous H₂S [92]. During artificial insemination, freezing thawing may cause oxidative stress damage to sperm [128]. Studies have validated that H₂S donors can protect sperm from oxidative stress damage in vitro, maintain sperm motility, and reduce acrosome loss [93]. Notably, high levels of H₂S can reduce sperm migration [129]. On the contrary, the co-administration of low-concentration H₂S donors and NO donors can enhance the forward motility of boar sperm and protect the integrity of the plasma membrane under oxidative stress [130]. It was found that H₂S-releasing agents based on amino acids simulated the release of physiological levels of H₂S in the presence of carbonic anhydrase without exerting harmful effects on cell function. In addition, the H₂S-releasing agents enhanced the forward motility of sperm during short-term treatment, which may prolong the survival of sperm [131]. These results suggest that the regulatory effects of H₂S on sperm motility in vitro may contribute to assisted reproduction.

In addition, the contraction of the epididymis and vas deferens promotes the expulsion of sperm. H₂S has been shown to regulate the function of the vas deferens and epididymis. It relaxes the smooth muscle of the vas deferens in a concentration-dependent manner and participates in the regulation of tonic contraction of the vas deferens [23,24]. Li et al. [24] used NaHS to treat vas deferens strips pretreated with L-NAME, TEA, iberiotoxin, GLB, 2-aminoethoxydiphenylborate (2-APB, a transient receptor potential channel inhibitor), and apamin (a small conductance calcium-activated potassium channel blocker). The results showed that L-NAME, GLB, 2-APB, and apamin did not affect the response of the vas deferens to NaHS, whereas TEA and iberiotoxin counteracted the relaxation effects of NaHS, suggesting that H₂S targets BKCa channels to regulate vas deferens relaxation. Furthermore, N-ethylmaleimide, a sulfhydryl alkylation compound that protects thiols from oxidation, prevented the NaHS-induced relaxation of the vas deferens smooth muscle. These findings suggest that H₂S directly or indirectly regulates the activity of BKCa channels through S-sulfhydration, thereby relaxing vas deferens smooth muscle. The microenvironment of the epididymal lumen is conducive to maintaining sperm motility, and functionally mature sperm are stored in the tail of the epididymis before ejaculation to maintain their immobility [132]. H₂S promotes K⁺ secretion in the rat epididymal epithelium by activating K_ATP and BKCa channels. High K⁺ concentrations in the luminal fluid of the cauda epididymis can inhibit sperm motility in a pH-independent manner. H₂S promotes the formation of an epididymal luminal microenvironment with high K⁺ concentrations, thus maintaining the cauda epididymal sperm in a quiescent state before ejaculation [56]. These results suggest the involvement of H₂S in ejaculation. Additional research could help in creating new treatment approaches for asthenospermia, spermatorrhea, and premature ejaculation.

4.4. H₂S Relieves Testicular Damage

H₂S has been discovered to suppress the expression of iNOS and TNF-α, an inflammatory cytokine, to reduce tissue damage caused by inflammation, consequently alleviating I/R injury in testicular torsion. In addition, H₂S inhibited apoptosis by suppressing the expression of apoptotic protease activator factor-1 (Apaf-1) [90]. These findings suggest that H₂S plays a protective role in testicular injury. Furthermore, H₂S has been shown to alleviate varicocele-induced testicular and epididymal damage. It was found that the weight of the left testis and epididymis and the diameter and epithelial thickness of seminiferous tubules were significantly reduced in rat models of varicocele. Long-term administration of NaHS restored these parameters by reducing oxidative stress and apoptosis in the testis [133]. Similarly, GYY4137 has been shown to alleviate varicocele-induced ipsilateral epididymal injury by activating the PI3K/Akt pathway in rat models (Figure 5) [94]. In addition, the researchers found that co-treatment with testosterone and NaHS reduced the damage caused by varicocele in rats. Compared with long-term administration of NaHS, combination therapy reduced the duration of treatment and the administered dose [134].

5. Effects of H₂S on the Female Reproductive System Diseases

5.1. H₂S Relieves the Preterm Birth

5.1.1. H₂S Relieves Abnormal Uterine Contractions-Caused Preterm Birth

H₂S participates in the regulation of uterine contractility by relaxing the myometrium. Sidhu et al. [25] found that both endogenous H₂S-generated substrates L-Cys and exogenous H₂S donors NaHS effectively reduced the spontaneous contractions of pregnant rat uterine muscles in a concentration-dependent manner in vitro. It has consistently been shown that H₂S exposure during gestation from day 6 prolongs labor in Sprague-Dawley rats [135]. In pregnant humans and rats, it was demonstrated that administration of GYY4137 or NaHS prevented preterm birth by relieving spontaneous contractions in the myometrium as well as oxytocin stimulation [136].

Sildenafil, a selective inhibitor of phosphodiesterase type 5 (PDE5), relaxes the smooth muscles of the pregnant uterus; however, its exact mechanism of action remains unclear. Mitidieri et al. [137] showed that sildenafil significantly increased the production of H₂S in the mouse uterus, whereas CSE inhibitors attenuated the effects of sildenafil. However, sildenafil did not increase H₂S production in CSE^-/- mice. Likewise, CSE^-/- mice showed less inhibition of spontaneous uterine contractility by sildenafil. These results suggest that the CSE/H₂S system regulates the effects of sildenafil and the contractility of the uterus in mice. Given the therapeutic effects of H₂S against uterine contractility disorders, H₂S can be used as a novel inhibitor of premature uterine contractions to prevent preterm birth [138].

5.1.2. H₂S Relieves Inflammation-Caused Preterm Birth

Research conducted in the past has indicated that infection or inflammation is linked to 40% of premature births [139]. Activation of the maternal immune system results in elevated levels of white blood cells and inflammatory substances in the uterine muscle, which stimulate uterine contractions, cervical ripening, and rupture of the fetal membranes, ultimately causing premature birth [140]. Chen et al. [96] found that the expression of NLRP3, TLR4, and activated NF-κB was upregulated in mouse models of LPS-induced preterm uterine contractions. Treatment with NaHS decreased the expression of these three inflammatory factors and uterine constriction-associated protein (CAP) in a dose-dependent manner, consequently delaying LPS-induced preterm delivery in mice. However, treatment with TAK-242 (a TLR4 inhibitor) and BAY11-7082 (an NF-κB inhibitor) counteracted the increase in NLRP3 expression in human uterine SMCs treated with IL-1β. It is suggested that H₂S suppresses the activation of NLRP3 inflammasomes through restraining the TLR4/NF-κB pathway, thereby maintaining the quiescent state of the uterus during infection or inflammation [97]. Upon binding to TLR4, LPS activates various signaling pathways, such as extracellular signal-activated kinase 1/2 (ERK1/2) and NF-κB [141]. Liu et al. [97] demonstrated that exposure to LPS led to elevated phosphorylated ERK1/2 and p65 levels in the myometrium, along with increased leukocyte infiltration into intrauterine tissue and upregulation of pro-inflammatory cytokines like IL-1β, IL-6, TNF-α, CCL2, and CXCL15. However, administration of NaHS counteracted the effects of LPS, indicating that H₂S could be a potential focus for addressing infection-related preterm labor (Figure 6).

5.2. H₂S Promotes the Endometrial Angiogenesis

During the proliferative phase of the menstrual cycle and during pregnancy, angiogenesis is responsible for the regeneration of the endometrium and the expansion of large blood vessels at the maternal-fetal interface [142,143]. Studies have shown that CBS expression and H₂S production increase in the endometrium during pregnancy and the proliferative phase of the menstrual cycle. This increase is positively correlated with an increase in endogenous estrogen levels and endometrial angiogenesis during the two periods [64]. When the menstrual cycle is in the proliferative phase and pregnancy is in full swing, estrogen levels are remarkably high [144]. In ovariectomized nonpregnant ewes, estradiol (estradiol-17β, E2) replacement therapy enhances the CBS levels in the uterine artery endothelium and smooth muscle while having no effect on CSE expression [98]. In addition, E2 promotes CBS and CSE expression in ewe uterine artery SMCs (UASMCs) in a manner that depends on time and concentration. Notably, agonists that target specific estrogen receptors, namely, ERα or ERβ, can promote the expression of CBS and CSE in UASMCs [99]. Qi et al. [64] found that E2 increased the production of H₂S by stimulating the estrogen receptor-dependent selective upregulation of CBS in endometrial stromal cells (ESCs). Nevertheless, the precise function of estrogen receptors in controlling the transcription of CBS is still not fully understood. Bai et al. [145] showed that E2 promoted CBS expression and H₂S production in non-pregnant and pregnant uterine artery endothelial cells by activating the CBS initiation program. The binding of ERα and ERβ to estrogen response elements in the CBS promoter region plays an important role in this process. Therefore, ERα or ERβ activators can stimulate CBS/H₂S production to levels comparable to E2-stimulated levels, whereas monotherapy with ERα or ERβ antagonists may block the E2-stimulated response. E2 has the ability to promote angiogenesis in endometrial microvascular endothelial cells (EMECs) when co-cultured with ESCs, leading to proliferation, migration, and tube formation. This phenomenon is validated by the higher potency of proliferating endometrium-derived ESCs than secretory endometrium-derived ESCs in stimulating the migration of EMECs. H₂S donors can significantly stimulate angiogenesis in EMECs, whereas downregulation of CBS but not CSE inhibits the migration of EMECs [64]. Altogether, as the human endometrium proliferates and as pregnancy progresses, the CBS/H₂S system is upregulated and participates in the regulation of estrogen during endometrial vascular remodeling. Various reproductive diseases, like endometriosis, are linked to the abnormal growth of blood vessels in the endometrium [146]. Research has revealed elevated levels of both CBS and CSE in the abnormal endometrial tissues of individuals and mice suffering from endometriosis [100]. However, inhibition of CBS and CSE notably reduced the number and weight of endometrial lesions in an allograft mouse model of peritoneal endometriosis. In vitro experiments showed that both exogenous and endogenous H₂S promoted the proliferation of human ESCs, which was attenuated by inhibitors of CBS, CSE, or NF-κB (Figure 6). The findings indicate that H₂S promotes the proliferation of ESCs via the triggering of the NF-κB pathway, offering valuable insights for the potential application of H₂S inhibition in treating endometriosis.

5.3. H₂S Promotes Embryo Implantation

The effects of H₂S on uterine function are negligible. Dorman et al. [43] showed that continuous exposure to H₂S (10–80 PPM, 6 h/d) before and after reproduction in female Sprague-Dawley rats (F0 generation) had no significant effect on pregnancy success rates, pregnancy process, or litter size. Guzman et al. [101] showed that the uterus of CBS-knockout pregnant mice underwent significant morphological changes and had decreased quality. Although the number of fertilized eggs implanted in the uterus did not decrease, the embryo survival rate decreased significantly. Notably, neither the ovaries nor the ovulatory oocytes of CBS-knockout mice showed significant morphological changes, and transplantation of CBS-null ovaries into mice with ovariectomizing mice fully restored fertility. These findings suggest that uterine dysfunction instead of ovarian dysfunction results in sterility in CBS-deficient female mice. The dynamic regulation of the uterine fluid environment required for embryo implantation is inseparable from the ion transport of the endometrial epithelium [147]. Cystic fibrosis transmembrane conductance regulator (CFTR) facilitates the release of Cl^- to promote the secretion of uterine fluid; however, its overexpression can lead to the formation of hydrops and hence prevent embryo implantation [148]. Xu et al. [107] showed that exogenous administration of H₂S increased the concentration of Cl^- in mouse endometrial epithelial cells and the I^--dependent short-circuit current (ISC). A specific CFTR inhibitor, CFTRinh-172, attenuated the increase in ISC following pretreatment. In addition, disruption of endogenous H₂S synthesis impaired embryo implantation. The findings indicate that H₂S suppresses the secretion of transepithelial anions in mouse endometrial epithelium by blocking CFTR during early-stage pregnancy, thereby regulating the uterine fluid volume to prepare for embryo implantation.

5.4. H₂S Promotes Postpartum Myometrial Recovery

During pregnancy and childbirth, the uterus undergoes molecular and functional changes that affect the regulation of H₂S. It was shown that L-Cys decreased the extent of natural constrictions in non-laboring or laboring myometrial tissues in a dose-dependent manner, according to You et al. [95]. Elevated levels of L-Cys heightened the frequency of natural uterine constrictions and triggered sustained constrictions. GLB pretreatment attenuated the suppressive impact of L-Cys on the strength of natural contractions in myometrium strips, indicating that L-Cys hinders uterine muscle constrictions through the stimulation of K_ATP channels. Compared with non-laboring myometrial tissues, laboring myometrial tissues had decreased CBS and CSE levels. In addition, the amplitude of spontaneous contractions and baseline muscle tension were less affected by L-Cys in laboring myometrial tissues. These findings suggest that decreased levels of H₂S contribute to the transition of the uterus from quiescence to contraction after parturition. Therefore, H₂S can be used to prevent infection-related preterm births and treat endometriosis. Overall, H₂S has a beneficial impact on controlling biological functions such as angiogenesis in the proliferative phase of the female menstrual cycle and pregnancy; the formation of a favorable uterine fluid microenvironment for embryo implantation; and the recovery of myometrium after delivery, which is important for maintaining the integrity of uterine structure and function.

5.5. H₂S Improves Pre-Eclampsia

Placental abnormalities can lead to severe complications like pre-eclampsia and restricted fetal growth. The regulatory effects of H₂S on the placenta are involved in the pathogenesis of pre-eclampsia. Sarno et al. [102] indicated that the generation of H₂S in postpartum placental models of early-onset and late-onset pre-eclampsia was comparable to that in the placenta of pregnant women without health issues. Following the addition of L-Cys, the late-onset pre-eclampsia group exhibited increased H₂S production compared with the normal pregnancy and early-onset pre-eclampsia groups. Nevertheless, research has indicated that the mRNA levels of CBS are lower in placental models of early-onset pre-eclampsia compared with placentas from healthy pregnant individuals, while CSE expression remains unchanged. The decreased mRNA levels of CBS result in reduced H₂S production [58]. The levels of CSE/H₂S are lower in the plasma and placental tissues of pregnant women with pre-eclampsia than in those of healthy pregnant women. Notably, the decreased expression of H₂S and CSE leads to maternal hypertension and placental abnormalities [60]. Although these findings are contradictory, they suggest the potential involvement of the L-Cys/H₂S pathway in the pathogenesis of pre-eclampsia. One of the major factors involved in the development of pre-eclampsia is the increased expression of soluble FMS-like tyrosine kinase 1 (sFlt-1) and soluble endocrine hormone (sEng) in the placenta [149]. Studies have shown that CSE knockdown increases the release of sFlt-1 and sEng from human umbilical vein endothelial cells, whereas its overexpression inhibits the release of the two factors. Moreover, treatment with GYY4137 can also inhibit sFlt-1 and sEng at the circulating level [60,150]. Increased sFlt-1 expression has been strongly associated with increased levels of metalloproteinase 10 (ADAM10). ADAM10 levels are higher in placenta samples from women with pre-eclampsia than in those from healthy women and are negatively correlated with the levels of CBS and CSE. Silencing of ADAM10 leads to decreased sFlt-1 release, while administration of NaHS and L-Cys effectively suppresses ADAM10 expression [103]. Hu et al. [59] showed that when syncytiotrophoblasts obtained from the placentas of both healthy women and women with pre-eclampsia were exposed to NaHS and L-Cys in culture, the protein expression of sFlt-1 was decreased, the half-life of sFlt-1 mRNA was shortened, and the expression of miR-133b targeting sFlt-1 was increased. These results suggest that H₂S delays the development of pre-eclampsia by suppressing the release of sFlt-1. The progression of pre-eclampsia is driven by TLR4-mediated sympathetic hyperactivity caused by inflammation of the rostral ventrolateral medulla oblongata (RVLM). Research [104] indicated elevated plasma levels of inflammatory markers and norepinephrine, along with decreased levels of H₂S, in pre-eclampsia patients compared with healthy pregnant individuals. Similarly, compared with normal rats, rats with deoxycorticosterone acetate-induced pre-eclampsia exhibited higher renal sympathetic activity, higher plasma norepinephrine levels, and lower H₂S levels in RVLM. Injection of the TLR4 agonist LPS induced microglia-mediated inflammation in RVLM, increased the sympathetic tone, and aggravated pre-eclampsia-like symptoms in pregnant rats. However, administration of NaHS alleviated these manifestations in both rats with PE and pregnant rats treated with LPS. These findings suggest that H₂S reduces the severity of pre-eclampsia by inhibiting TLR4 and attenuating inflammatory responses in RVLM, providing a new target for the treatment of pre-eclampsia. Placental health relies on endogenous H₂S, and decreased H₂S levels in vivo may contribute to the development and progression of pre-eclampsia.

Pre-eclampsia is closely associated with an imbalance of angiogenic growth factors and is characterized by an antiangiogenic state. Consequently, the pro-angiogenic effects of H₂S on the placenta may contribute to the alleviation of pre-eclampsia. H₂S-miRNA has been reported to regulate vascular endothelial growth factor (VEGF) in the placenta and promote placental angiogenesis, thereby ameliorating pre-eclampsia [105]. CBS, CSE, VEGF, miR-200c, miR-20a, and miR-20b are downregulated in placentas during pre-eclampsia. In a study, treatment of explants and trophoblasts isolated from healthy placentas with H₂S donors and L-Cys elevated the protein expression of VEGF, prolonged the half-life of VEGF mRNA, and decreased the expression of miR-200c, miR-20a, and miR-20b. Unlike the mimics and inhibitors of miR-200c, those of miR-20a or miR-20b affect VEGF expression at the protein level but not at the mRNA level [105]. Chen et al. [106] showed that HTR-8/SVneo cells from villous trophoblasts increased the migratory capacity of sheep placental arterial endothelial cells (oFPAECs), while the use of CBS inhibitors reduced this impact. In oFPAECs, the effects of H₂S donors on angiogenesis were comparable to those of VEGF; however, the rapid activation of eNOS, Akt1, and ERK1/2 upon H₂S donor phosphorylation was slightly weaker than that upon VEGF phosphorylation. ERK1/2, PI3K/Akt1, and eNOS/NO are key signaling pathways that mediate angiogenesis. When activated by H₂S donors, specific PI3K inhibitors can block the phosphorylation of Akt1 and eNOS without affecting ERK1/2. Similarly, ERK1/2 inhibitors do not affect the phosphorylation of Akt1 and eNOS. These findings suggest that H₂S derived from trophoblasts stimulates placental angiogenesis through phosphorylation of the PI3K/Akt1/eNOS and ERK1/2 pathways in endothelial cells. Decreased expression of placental growth factor (PIGF) indicates impaired placental angiogenesis. In vitro studies have shown that PAG decreases the production of PIGF and hinders the invasion of trophoblasts in human placental explants in the initial stages of pregnancy. PAG can lead to high blood pressure and liver impairment in pregnant mice, promote the development of abnormal labyrinth vessels in the placenta, and cause fetal development restriction (Figure 7). However, treatment with GYY4137 counteracts the effects of PAG [60,150]. Consequently, dysfunction of the CSE/H₂S system may lead to placental abnormalities in pre-eclampsia and affect normal fetal development.

5.6. H₂S Improves Fetal Growth Restriction

Impaired blood flow in the umbilical artery and elevated placental vascular resistance are frequently linked to intrauterine growth restriction (IUGR) [151]. In addition, pre-eclampsia limits fetal growth and development in utero. It was demonstrated that the expression of CSE was notably lessened and that of miR-21, a negative regulator of CSE expression, was markedly elevated in IUGR and pre-eclampsia with abnormal Doppler umbilical artery waveform (PE-AD) placentas than in healthy placentas [61]. The same expression patterns were observed in villus explants subjected to hypoxia-reoxygenation damage and placental stem villus arteries (SVAs) [61,108]. The smooth muscle phenotype of SVAs is in a state of dedifferentiation, with decreased expression of SMC differentiation markers such as myosin heavy chain, smooth muscle actin, and desmin and increased expression of dedifferentiation markers such as retinol-binding protein 1 and MMP-2. Notably, under normoxic conditions, PAG results in a dedifferentiated state of SMCs in SVA tissues ex vivo, whereas treatment with the H₂S donor diallyl trisulfide reverses the effects of PAG. Lu et al. [108] found that neonatal birth weight had a positive correlation with the expression of CSE and SMC differentiation markers but a negative correlation with SMC dedifferentiation markers in placental tissues. Consistently, placental SVAs at IUGR had a smaller lumen diameter and unchanged wall thickness than at term delivery or preterm labor and were also associated with the loss of end-diastolic flow. These findings suggest that IUGR is involved in the vascular remodeling of SVAs induced by abnormal CSE/H₂S signaling. NaHS relaxes blood vessels in normal placentas pre-constricted with the thromboxane A2 mimetic U46619 in a dose-dependent manner (Figure 7). These relaxant effects of NaHS can be reversed by GLB and L-NAME [61]. Therefore, H₂S donors may reduce placental blood flow obstruction by preventing the dedifferentiation of SMCs and dilating blood vessels through the K_ATP channel and NO/cGMP pathway, which may enable normal fetal growth and development.

5.7. H₂S Alleviates Recurrent Spontaneous Abortion

Immune homeostasis is often dysregulated in unexplained recurrent spontaneous abortion (URSA) [152]. H₂S can regulate immune homeostasis by promoting the differentiation of regulatory T cells (Tregs), thus helping the fetus to escape the attack of the maternal immune system on paternal antigens [153,154]. Wang et al. [109] showed that treatment of decidual tissues from pregnant mice with AOAA or PAG significantly reduced the production of TGFβ, a crucial factor for the growth, specialization, and cell identification abilities of Treg cells [155]. However, treatment with GYY4137 or NaHS effectively restored the expression of TGFβ. At the time of embryo implantation, destruction of the luminal epithelium initiates the inflammatory response, and the maternal immune system is reprogrammed from the T-helper 1 (Th1) state to the Th2 state to ensure immune tolerance during pregnancy [156,157]. In a study, the decidua of resorbed fetuses exhibited increased levels of Th1 cytokines (IFN-γ and TNF-α) and decreased levels of Th2 cytokines (IL-4 and IL-6), validating the relationship between immune homeostasis and URSA. CBS/H₂S in HTR8/SVneo cells could potentially suppress NF-κB signaling pathway activation by decreasing IL-1R1 levels, leading to decreased expression of inflammatory response factors like COX2 and reduced PGE2 secretion [109]. Abnormal expression of PGE2 and COX2 is the cause of URSA [158,159]. In early human pregnancy, H₂S regulates three immune tolerance-related proteins, namely, human chorionic gonadotropin, diamide-2,3-dioxygenase [160,161,162,163,164], and thymic stromal lymphopoietin [165], which maintains the Th2 state in the decidua. Inadequate trophoblast invasion may lead to pregnancy loss in the early stages [166]. CBS expression is decreased in placental villous cytotrophoblasts in URSA compared with normal early pregnancy, while CSE expression remains unchanged. Pregnant mice lacking CBS or treated with AOAA show a notable rise in embryo resorption rate and percentage of embryo loss, both of which can be decreased effectively with GYY4137 or NaHS. Exogenous administration of H₂S enhances the migratory and invasive abilities of HTR8/SVneo and JEG3 cells (human placental trophoblasts), along with increasing the levels of MMP-2 and VEGF proteins [109] (Figure 7). Overall, placental CBS/H₂S signaling plays a crucial role in early pregnancy by regulating immune tolerance and trophoblast invasion.

5.8. H₂S Improves Placental Oxidative Damage

Various placental complications are associated with smoking during pregnancy or exposure to cigarette smoke during pregnancy [167,168]. Zhao et al. [110] showed that exposure of pregnant rats to cigarette smoke on days 7–20 of gestation increased the levels of 8-OHdG, DNA oxidative damage markers, and MDA and aberrantly decreased the levels of Nrf2 in the placenta, suggesting oxidative damage in the placenta. However, the administration of NaHS significantly counteracted these changes. Furthermore, it successfully decreased the placental redox imbalance caused by cigarette smoke by reinstating the overall antioxidant capacity, elevating the GSH/GSSG ratio, and boosting the functions of SOD, catalase, and glutathione peroxidase to prevent ROS generation. The results indicate that H₂S decreases the generation of ROS caused by cigarette smoke exposure in pregnant women by activating the Nrf2 pathway, ultimately reducing oxidative harm to the placenta.

The placenta is crucial in the pathogenesis of gestational diabetes mellitus (GDM) [169]. In GDM, deficiency of H₂S-producing enzymes in the placenta results in excessive activation of the NLRP3 inflammasome, an important inflammatory cytokine involved in the initiation of maternal insulin resistance [111]. Therefore, H₂S may be involved in the pathogenesis of GDM by controlling NLPR3 inflammasome activation in the placenta. Altogether, H₂S plays an essential role in promoting normal placental angiogenesis and blood flow, maintaining placental immune homeostasis in early pregnancy, and facilitating the growth and development of embryos after implantation. Dysfunction of the H₂S system is involved in pre-eclampsia, URSA, IUGR, and other placenta-related diseases caused by various factors, whereas H₂S supplementation plays an effective therapeutic role in these diseases.

5.9. H₂S Promotes Ovulation

Elevated homocysteine levels may affect oocyte quality or interfere with ovulation. CBS is the main enzyme involved in homocysteine metabolism and is widely distributed in the ovary. An impairment of CBS activity can lead to hyperhomocysteinemia [101]. Liang et al. [112] found that CBS levels were decreased in mouse ovarian granulosa cells, whereas homocysteine and methionine levels were notably increased in the follicular fluid. Furthermore, the rate of in vitro oocyte maturation was significantly reduced in the CBS-inhibited granulosa cell medium cultured oocyte model. CBS-knockout females have been shown to develop fewer follicles than wild-type female rats [101]. The results indicate that CBS is responsible for controlling oocyte maturation through the maintenance of homocysteine levels in the oocyte environment. Before ovulation, the expression of CSE in granulosa cells is increased by the upregulation of luteinizing hormone (LH), leading to higher levels of mRNA and protein. Administration of NaHS can enhance the mRNA levels of proteins related to cumulus expansion and follicle rupture, including amphiregulin, epiregulin, and plasminogen activator, in mouse granulosa cells, thus facilitating ovulation. However, CSE inhibitors can block ovulation [170]. Altogether, H₂S has positive effects on germ cell development in the ovary.

5.10. H₂S Regulates Oviductal Transport

The fallopian tube provides a favorable microenvironment for the final maturation of gametes, fertilization, and the primary stage of embryonic development [171]. However, if the embryo is retained in the fallopian tube, it can lead to an ectopic pregnancy. Typically, the fallopian tube’s smooth muscle contracts regularly like a sphincter in normal circumstances. Ning et al. [26] showed that administration of NaHS or L-Cys resulted in the relaxation of the smooth muscle in the fallopian tube in a manner that depended on the dosage. Additionally, there was a notable rise in the levels of CBS and CSE in the epithelial cells of the fallopian tube in cases of ectopic pregnancy. Treatment with an H₂S donor and a CBS activator impaired embryonic trafficking and induced asynchronous development in mice. In addition, the effects of a CBS inhibitor were reversed after administration of NaHS or GYY4137. These findings suggest that changes in H₂S signaling in the oviduct of pregnant mice delay embryo transport and early embryonic development, resulting in the failure of timely embryo implantation in the uterus. The normal transmission of H₂S signals is essential for the oviductal transport of embryos.

5.11. H₂S Modulates the Female Sexual Response

In addition, H₂S plays an important role in female sexual responses. Srilatha et al. [16] showed that NaHS significantly relaxed rabbit vaginal smooth muscle strips in a way that depended on the concentration; however, these relaxation effects were reversed after treatment with MDL-12,330a (an adenylate cyclase inhibitor), L-NAME, ODQ, or GLB. The results indicate that the relaxation properties of H₂S on rabbit vaginal smooth muscle are mainly related to cAMP, NO/cGMP, and K_ATP channels. Sun et al. [65] showed that treatment with NaHS increased vaginal lubrication, which was related to the H₂S-induced increase in the concentrations of K⁺ and Cl⁻ ions in rat vaginal fluid. Furthermore, treatment with NaHS initially decreased ISC but subsequently increased it in isolated rat vaginal tissues. The decrease in ISC was sensitive to K_ATP channel inhibitors, whereas the increase in ISC relied on Cl^- ions and could be attenuated by CFTR inhibitors. Both inhibitors prevented NaHS-induced vaginal lubrication in vivo. Given that CSE is the main enzyme expressed in the vaginal epithelium, the CSE/H₂S pathway is crucial in regulating the secretion of vaginal fluid.

Overall, the pathway for producing H₂S is crucial for the maturation of oocytes and ovulation, as well as the transportation and growth of early tubal embryos and the regulation of vaginal smooth muscle relaxation and vaginal fluid secretion. On the contrary, abnormal H₂S signaling can inhibit ovulation, delay embryonic development, and lead to vaginal dryness. These pathological conditions can be treated by the exogenous administration of H₂S donors.

6. Conclusions and Discussion

In conclusion, H₂S regulates both male and female reproductive systems and participates in physiological processes such as penile erection, testicular reproduction, spontaneous contraction of the vas deferens, ejaculation, ovulation, pregnancy, and postpartum recovery, thus maintaining the normal structure and function of reproductive organs. Abnormal H₂S signaling may lead to andrological conditions such as ED, male infertility, premature ejaculation, and spermatorrhea, as well as obstetric conditions such as embryo implantation failure, recurrent miscarriages, fetal growth restriction, pre-eclampsia, and premature delivery. The mechanisms through which H₂S promotes penile erection remain unclear. H₂S participates in muscle relaxation through activation of BKCa and Kv channels, inhibition of the RhoA/ROCK signaling pathway, and upregulation of cGMP in an NO-independent manner. Ghasemi et al. [50] found that H₂S relaxed the corpus cavernosum at pharmacological concentrations while inhibiting the relaxation of NO at physiologically relevant concentrations. Studies reporting similar findings are limited; therefore, further investigation is necessary. H2S has been shown to alleviate ED by inhibiting superoxide production in cavernosum and vascular SMCs [68]. Superoxide produced by NADPH oxidase (NOX) can reduce the bioavailability of NO by reacting with it to form reactive nitrogen species [172,173,174,175], increase cGMP metabolism by upregulating PDE5, and activate ROCK [176], eventually impairing erectile function. NO can inhibit the activity and expression of NOX through the cGPM-PKG pathway [177,178,179]; consequently, diseases or conditions characterized by impaired endothelial structure, such as diabetes, are predisposed to ED. The inhibitory effects of H₂S on superoxide production are complex. H₂S not only inhibits the activity and expression of NOX through the cAMP-PKA and cGMP-PKG pathways [180,181] but also regulates the expression of Nrf2 and its downstream anti-oxidative stress proteins, including SOD, NAD(P)H quinone oxidoreductase, and HO-1, through S-sulfhydration of Keap1, consequently suppressing superoxide production [182]. In addition, S-sulfhydration of H₂S is involved in the activation of K_ATP channels [183] and BKCa channels [24] and the reduction of PDE5A dimerization [184]. The mechanisms through which H₂S regulates female reproductive organs are complex and diverse but have not been investigated comprehensively. Relaxation of uterine vessels, placental vessels, and vaginal smooth muscle relies on K_ATP channels, which are associated with NO/cGMP signaling. In different environments, H₂S can regulate inflammation and cell proliferation by selectively activating or inhibiting ERK1/2 and NF-κB, alleviating pre-eclampsia and preventing infection-related preterm birth. With in-depth research on H₂S, various H₂S-based therapeutic strategies and H₂S-releasing drugs, including ZnS@TA [123], ACS6 [20], HS-NSAID [121], and H₂SDox [122], have been developed for the treatment of reproductive system diseases. However, studies investigating the biological role of H₂S in the reproductive system are fewer than those focusing on other human organ systems; moreover, a systematic description is lacking. Hence, further research is needed to explore the impact of H₂S on the reproductive system in order to enhance the efficacy of treatments for reproductive system disorders.