Hydrogen sulfide (H₂S) and implantation failure

In Assisted Reproduction Technologies (ART) practices we speak of implantation failure when a good quality embryo is transferred into the uterus but this fails to nestle in the endometrium. Failure to implant after several transfers of good embryos is referred to as repeated implantation failure. This is a serious form of infertility because there are few possible remedies.

Role of uterine contractility

A first obvious cause of failure to implant a good embryo is its expulsion into the vaginal canal by uterine contractions immediately after transfer. These contractions can be triggered by the transfer procedure itself if, by mistake, the uterine wall is touched and stimulated. The consolidated skill of the operators and the availability of precise ultrasound guides make this phenomenon rare. More often, it is the patient who has excessive uterine responsiveness and triggers improper contractions [Chung 2017].

Under physiological conditions, uterine contractility is kept quiescent starting from the moment of ovulation and up to delivery by the action of a gasotransmitter, hydrogen sulphide (H₂S) [You 2017]. This, defined as the natural tocolytic agent, is released under estrogenic stimulation by the granulosa and endometrial cells already before ovulation, after ovulation by the corpus luteum (LH-progesterone stimulus) and, after implantation by the placenta (hCG-progesterone stimulus). Only at the time of delivery there will be a drop in H₂S release so that labour contractions can start. Therefore, a deficient release of H₂S can result in an excessive reactivity of the uterus with expulsion of the embryo immediately after its deposit in the uterus.

Role of immunity

In human (sexual) reproduction, the embryo is formed by joining the maternal gamete with the paternal one, which is immunologically foreign. Therefore, both the embryo and the placenta are half foreign and should be attacked by the immune system.

This does not happen because the H₂S released from granulosa, corpus luteum and placenta induces a conversion of T lymphocytes into their regulatory form (Treg) [Yang 2015], that is, the one that blocks the aggression by the effector lymphocytes. Therefore, a lack of H₂S release can result in a lack of immune tolerance [Dilek 2020] in the endometrium with immediate aggression to the embryo even before it can implant itself.

Role of vascularization

Even if it has not been expelled by contractions and has not been attacked by the immune system, in order to implant, the embryo also needs to find a welcoming endometrium, capable of supplying it with everything it needs.
The endometrium is ready because already during the follicular phase of the previous cycle H₂S had begun to stimulate the growth of new small vessels (spiral arterioles) in the endometrium [Qi 2020Chen 2017]. These new vessels must also carry an abundant flow of blood and this too is guaranteed by H₂S which induces vasodilation of the uterine artery and then of the entire placental district [Li 2020].

H₂S is the physiological partner of implantation

In summary, the physiological release of H₂S intervenes in facilitating all the critical steps of embryonic implantation.

H₂S induces relaxation of the uterine muscles, preventing expulsive contractions and facilitating the practice of the transfer.

It also prepares the endometrium to be adequately immune tolerant and well-endowed with small arteries with good blood flow.

Pharmacological treatments

Given the role of uterine contractions in implantation failure, a first possible pharmacological remedy is the administration of muscle relaxants. However, a clinical study involving a potent muscle relaxant (atosiban) yielded no measurable effects.

The standard implant support treatment is instead luteal support with progesterone, vaginally or injected. Progesterone is in fact the hormone responsible for inducing immune tolerance as well as vascularization and it is believed that an excess of the same can be of help. However, progesterone deficiency has never been confirmed in patients suffering from implantation failure. Rather, as a result of pharmacologically induced multiple follicular development, they tend to have higher than normal progesterone levels.

The clinical efficacy of progesterone in support of the implant is clinically demonstrated, but it is modest. This, on the one hand confirms the importance of contractility and immunity in the implantation process, on the other it tells us that there must be other mechanisms involved. In fact, progesterone acts largely by triggering the release of H₂S: If this downstream mechanism does not respond, increasing the progesterone stimulus is of little benefit. The release of H₂S will remain modest and its effects will continue to be lacking.

Implantation failure, H₂S and diet

The reasons for a reduced efficiency of H₂S release are not known but certainly individual genetics, diet and their interaction are involved.

We produce H₂S both through enzymatic mechanisms and with spontaneous reactions that take place in the circulation. Both enzymatic and spontaneous reactions are strictly based on the availability of vitamin B6 in a dose-dependent manner: the higher the level of active B6, the more the release of H₂S increases. B6 is widely contained in fish, organ meats, potatoes and other vegetables, although mainly in an inactive form.

The food substrate for releasing H₂S is the amino acid cysteine, a normal constituent of food proteins, especially vegetables. The free -SH group of cysteine is precisely the one that, with the addition of a hydrogen (H), forms H₂S.

A varied and balanced diet does not struggle to provide us with all the B6 and cysteines we need. However, we are not all equally efficient at activating B6 while cysteines, even if well present in the diet, are primarily converted to other uses. Therefore, overall healthy but borderline balanced diets may not adequately support the release of H₂S if in the presence of weak individual genetics and/or increased demand.

It has recently been proven that a food supplementation with supra-physiological doses of activated vitamin B6 (pyridoxal 5-phosphate) together with cysteines (in the form of L-cystine) and taurine (which conveys the cysteines towards the production of of H₂S) is capable of measurably increasing the endogenous release of of H₂S [Dattilo 2022].

Recommended readings

• Chen D-B et al (2017). Human trophoblast-derived hydrogen sulfide stimulates placental artery endothelial cell angiogenesis. Biology of Reproduction, 2017, 97(3), 478–489
• Chung CHS et al (2017). The changing pattern of uterine contractions before and after fresh embryo transfer and its relation to clinical outcome. Reprod Biomed Online 2017; 34(3): 240-247
• Dattilo M et al (2022). Modulation of Human Hydrogen Sulfide Metabolism by Micronutrients, Preliminary Data. Nutrition and Metabolic Insights 2022; Volume 15: 1–13
• Dilek N et al (2020). Hydrogen sulfide: An endogenous regulator of the immune system. Pharmacol Res 2020; 161: 105119
• Li Y et al (2020). Hydrogen Sulfide Relaxes Human Uterine Artery via Activating Smooth Muscle BKCa Channels. Antioxidants (Basel) 2020 Nov 13;9(11):1127
• Qi Q-R et al (2020). Enhanced Stromal Cell CBS-H2S Production Promotes Estrogen-Stimulated Human Endometrial Angiogenesis. Endocrinology 2020; 161(11): bqaa176
• Yang R et al (2015). Hydrogen Sulfide Promotes Tet1- and Tet2-Mediated Foxp3 Demethylation to Drive Regulatory T Cell Differentiation and Maintain Immune Homeostasis. Immunity 2015; 43, 251–263
• You X et al (2017). Endogenous hydrogen sulfide contributes to uterine quiescence during pregnancy. Reproduction 2017: 153: 535–543

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