Neither rationale nor scientific evidence exist to support that double stimulation is potentially unsafe Free

Sir,

We have carefully read the opinion paper published by Dr Angelo Tocci about the hypothetical mechanism explaining the effectiveness of double stimulation in the same ovarian cycle (DuoStim) (Tocci, 2022). Tocci (2022) suggests that the higher oocyte number generally collected after the second stimulation is imputable to differentiation of two FSH-responsive stem cell populations, i.e. very small embryonic-like and human ovarian stem cells (OSCs), presumably present in the ovarian surface epithelium (OSE). The opinion viewpoint is that the first stimulation may trigger the commitment of oocyte progenitors to differentiate into mature oocytes. We certainly recognize the author’s efforts in explaining how anovulatory waves of follicle growth may result in oocytes like those collected after the first stimulation per embryological, clinical and perinatal outcomes (Vaiarelli et al., 2020). On the contrary, strong concerns arose about the misleading inference that a second stimulation is ‘potentially unsafe’. In fact, we see no biomolecular rationale to support the author’s conclusion.

The extraction of concepts suggesting pro-tumorigenic roles exerted by FSH in the context of DuoStim from in vitro studies and animal models, undermines the basis of trophic hormones endocrine functioning and is very far from being representative of human physiology. We have summarized our concerns hereafter.

First, the authors describing OSCs stated they are relatively rare (White et al., 2012) and, to the best of our knowledge, the physiological role of these cells, if any, was only supposed and never demonstrated in humans. To date, data supporting the role of OSCs potentially lead to exciting perspectives but are strictly confined to animal models transplanted with engineered cells or to specific in vitro systems not reproducible in vivo by an ovarian stimulation protocol. Indeed, OSC differentiation to an oocyte embedded into a primary follicle takes time, especially from primary to the FSH-sensitive phase: it certainly does not fit within the DuoStim timespan. These concepts are fully supported by a lesson from human physiology: no OSC differentiation occurs in post-menopausal women, nor in patients affected by FSH-secreting adenoma, where high serum FSH levels persist for years. Taken together, these considerations lead to the obvious conclusion that, if oocyte progenitors truly exist in humans, a second stimulation in one ovarian cycle cannot trigger stem cell differentiation.

Second, the existence of functional FSH receptors (FSHRs) in human pre-antral ovarian cells is another unanswered assumption missing full scientific support. Even assuming they exist, their physiological relevance is not supported by any evidence. Data were provided from multi-ovulatory species like rodents, and sheep as well, but findings in humans are weak and perhaps biased by methodological issues (e.g. unspecific staining methods, lack of proper experimental controls, use of genetically engineered cells and missing functional significance of mRNA transcripts) (Chrusciel et al., 2019).

Third, this opinion paper is characterized by weak arguments about the endocrine regulation of gonadotropin physiology. It is well-known that FSH downregulates FSHR mRNAs, which are replaced by LH/choriogonadotropin receptor (LHCGR) transcripts. In fact, after dominant follicle selection during the last stage of folliculogenesis, FSHR expression is inhibited (Jeppesen et al., 2012). Therefore, prolonged FSHR expression in the ovary is hardly possible under physiological and para-physiological conditions because it would lead to cell death and follicular atresia (Casarini et al., 2020). The replacement of FSHR by LHCGR, accompanied by the action of estrogens, is in fact a prerequisite to sustain the mitogenic signals supporting follicular cell growth. To date, no reliable steroidogenic human cell line permanently expressing FSHR exists, because the activation of apoptotic signals leads to cell colony loss.

Fourth, Tocci (2022) mentioned a list of FSH-dependent intracellular signaling pathways, genes associated with cell proliferation and differentiation, as well as the presence of cancer membrane markers, to support a supposed sensitivity of the OSE to the alleged tumorigenic activity of gonadotropins. We honestly think this is a mere speculation that leads readers through several, weakly associated mechanisms with no or largely limited scientific support. Most importantly, this opinion paper seems to ignore the obvious difference between mitogenic signals and oncogenic events. Whilst, on the one hand, certain cancer cells display hormone receptors because of a clear dysregulation in gene expression, on the other hand, tumor cell markers may be upregulated in healthy cells under certain physiological conditions controlled by hormones (e.g. gametogenesis, trophoblast invasion, placentation, etc.). The physiological upregulation of proliferative signals does not suggest in any way that gonadotropins exert tumorigenic effects. Decades of ART clearly agree with this view: there is no association between controlled ovarian stimulation and cancer. Claimed mild cancer risk increase in IVF patients, compared to general population, is reasonably biased by the condition of nulliparity and infertility per se (Rizzuto et al., 2019). We see neither reason nor scientific rationale why things should go differently when FSH is administered in the context of DuoStim.

In current ART, DuoStim figures amongst the most intriguing unconventional stimulation strategies to fully personalize the treatment in poor prognosis patients at high risk of discontinuation. The retrieval of competent oocytes from otherwise anovulatory waves of follicle development unveiled the extreme dynamisms of human folliculogenesis, thereby subverting historic dogmas and fully interpreting the empirical nature of IVF approaches. In this scenario, basic research is needed to discover mechanisms governing these events, and it certainly represents an exciting challenge with immediate clinical relevance. There is absolutely neither reason nor evidence to hypothesize safety issues of two back-to-back stimulations performed in a single ovarian cycle and the speculative inferences made by Tocci (2022) certainly do not support any concern. In our view, there is only one concept we support in the opinion, which is the need for a follow-up of treated patients. Yet this need refers to each and every strategy or technique implemented in ART and in medicine in general. We hope that more scientific evidence and less speculations will be produced in the future to move this field forward.

Conflict of interest

The authors declare that there are no conflict of interest to declare.

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