The mechanism for menstrual irregularities after COVID vaccination? Not really.

One of the oldest antivaccine tropes out there is that vaccines somehow affect fertility, especially female fertility. It’s a trope that dates back decades, starting at least as far back as the 1990s, when the myth surfaced in some countries in Africa and other underdeveloped nations that tetanus vaccines administered to young women would cause infertility, even “sterilize” them. The myth was particularly pernicious in Kenya, where Catholic bishops promoted it vigorously against all evidence, and, even though the medical establishment there called it out as nonsense, it was, like many such myths, widely believed because a source believed to be authoritative (bishops in the Catholic Church and some Catholic physicians) was promoting it. Unsurprisingly, HPV vaccines have also been a favorite target of this trope. Unsurprisingly, almost at the same time as mRNA-based COVID-19 vaccines received emergency use approval (EUA) in the US, the myth resurfaced, applied to the new vaccines. (Actually, it first resurfaced during the clinical trials for the Pfizer and Moderna vaccines.) They even came up with dubious scientific “mechanisms.”

Of course, we now know that the vaccines have been associated with transient menstrual irregularities, but not infertility. Not that that has stopped antivaxxers from continuing to claim horrific effects on fertility due to COVID-19 vaccines. So it was when the other day I saw Nicole Shanahan, running mate of longtime antivax activist turned improbable independent Presidential candidate Robert F. Kennedy, Jr., posted this on X, the hellsite formerly known as Twitter:

Study: https://t.co/6GbbL7qZ0R

— Nicole Shanahan (@NicoleShanahan) July 1, 2024

Notice how Shanahan claims that “they”—yes, “They”— “dismissed reports of unexpected bleeding and heavier flow following vaccination, claiming there was no data to support these links” and claim this as an example of “how the media and government often label an inconvenient truth a ‘conspiracy theory’ when it contradicts their predetermined narrative.” In actuality, scientists looked into anecdotal reports of menstrual irregularities associated with COVID-19 vaccines. That’s how we now know how transient such abnormalities are and that they do not affect overall fertility. That’s because the key to a good conspiracy theory is that there must be a coverup, and “They” must not want you to know what she is telling you.

But what about the study itself?

The first thing that I noticed was that this is primarily an in vitro study, using cultured human ovarian granulosa cells, which are the endocrine cells of the ovary, producing estrogen, progesterone, and other hormones that support ovarian function regulating the menstrual cycle and pregnancy. The funny thing is, too, that while there are some (very) preliminary clinical data presented, those data don’t really support the overall hypothesis. But what is the hypothesis? Specifically:

We hypothesized that the menstrual changes in post-vaccinated women may result from a direct effect of the vaccine on the GCs, leading to modifications in the expression and secretion of follicular hormonal regulators and thus affecting the cycle. InhibinB is produced primarily by GCs of FSH-dependent growing antral follicles and secreted predominantly during the follicular phase of the cycle (before ovulation)²⁷. There is little evidence of a correlation between serum levels of InhibinB and menstrual cycle parameters such as flow and bleeding duration. However, as InhibinB is a pivotal participant in the HPO feedback loop^24,25, that is correlated with cycle length²⁸, we focused on it.

So wait a minute. I might not be an OB/GYN or a scientist studying the endocrine or female reproductive systems, but I am a scientist and a molecular biologist. Right here, the authors seem to be saying that, even though there is little evidence of correlation between serum levels of what they looked at (InhibinB) and menstrual flow and bleeding duration, they decided to look at it anyway because “InhibinB” is a “pivotal participant in the HPO feedback loop^24,25, that is correlated with cycle length”? Maybe I’m wrong, but that seems like a bit of a stretch, especially given how little of the vaccine actually leaves the site of injection. The authors even admit earlier in the introduction that menstrual irregularities have been associated with other vaccines that most definitely do not leave the site of injection even to the very limited extent that mRNA-based COVID-19 vaccines do—and that other studies have suggested that the menstrual changes associated with COVID-19 vaccination are likely due to the immune response:

A link between menstrual irregularities and vaccine administration was demonstrated as early as 1913, tracking the typhoid vaccine²; since then, it has been shown that hepatitis B and papilloma viruses vaccines affect the menstrual cycle as well^2,4,8,13,14. It was suggested that this side effect is a result of the immune response to the vaccine, activation of endometrial immune cells that regulate the generation of the endometrium, or immunological influence on the secretion of follicular stimulating hormone (FSH) and luteinizing hormone (LH)^4,9,15. Examination of the COVID-19 vaccine pointed to an immunologic response, manifested by cytokine release (as Interleukin-8; IL-8)¹⁶.

Nevertheless, they trudge onward:

However, a direct effect of the vaccine on the ovary should be examined, as recent papers showed that ovarian cells could be directly infected by the SARS-CoV-2 virus¹⁷, and that the vaccine caused a biological response similar to that of prolactin signaling, which may lead to short-term menstrual irregularities¹⁸.

Of course, they justify this approach by referring to a Japanese biodistribution study that I’ve discussed at length before. If you want the details, here they are, but one key thing to note is that the biodistribution study used a dose of lipid nanoparticle ~18-35x higher by weight than the typical adult human dose from vaccine—as biodistribution studies tend to do in order to map where even the tiniest amount of drug or vaccine go in the body, which in this case was only around 0.1% of the vaccine dose of lipid nanoparticles going to the ovaries. The report to the FDA actually noted that less than 0.095% of the dose of radiolabeled lipid nanoparticles injected intramuscularly wound up in the ovaries.

So what did the authors do? Basically, they took cultured human primary granulosa cells (hpGCs) isolated from women 20–45 years of age with normal BMI who were undergoing IVF treatments in Sha’are Zedek Medical Center, Jerusalem between 11/2021 and 12/2022, noted to be “several months after the administration of the first and second doses of the Pfizer-BioNTech’s COVID-19 vaccine in Israel.” One could note here that these ovarian cells were not even “unvaccinated,” so to speak. Now let’s look at the actual methods by which these cultured cells were exposed to the vaccine:

The following day, hpGCs were washed with fresh culture medium supplemented with the Comirnaty® vaccine for COVID-19 (Pfizer). The pre-clinical trial published in the “Final Report”²⁰ by Acutias followed the distribution and accumulation of a LNP vehicle in the body of research-model rats after administration of a single dose, along 48 h²⁰, showing that outside the injection site, it was accumulated in 4 out of 28 examined organs of the rats, ovaries being one of them ( ~ 0.1%). In humans, adults are treated with a 30 µg dose/person (equivalent to a ~ 0.06 µg/ml concentration, theoretically distributed in an average 5 L blood volume for the adult women). Thus, we examined two vaccine concentrations (diluted from the original undiluted vile – “stock concentration” of 50 µg/ml): (i) 0.05 µg/ml (“injected dose”), to evaluate cell toxicity. (ii) 0.05 pg/ml (“end-organ dose”), representing the accumulated concentration in women’s ovaries (~0.1% of the injected dose).

Wait, what? They assumed that the 30 µg dose of vaccine distributed equally throughout the entire 5L blood volume of a typical adult woman? That’s one hell of an assumption, given that the vaccine is injected intramuscularly and is know to stay largely in the area. My initial reaction was also that we don’t inject the vaccine into the ovaries, but that was more my sarcasm coming out after seeing this study promoted by the likes of Nicole Shanahan. In reality, I will give the authors a modicum of credit for doing the alleged “end organ dose” of roughly 1:1,000 of the “injected dose.” Even so, though, given how much I question the very assumption that the entire vaccine dose distributes to the entire blood volume of an individual receiving the vaccine, I must point out that that dubious assumption applies even to the “end-organ” dose, which is based on the same assumption and just uses a concentration that is 1:1,000 less than the “full” dose. Because maximal accumulation of lipid nanoparticles in the rat ovaries occurred between 6-48 hours, that’s the time frame the authors examined:

We used this stock for the treatments as follows:

• “Injected dose”: 1 µl of the vaccine undiluted stock (50 µg/ml) in 1 ml of the cells culture medium results in a concentration of 0.05 µg/ml. This concentration resembles the concentration of the vaccine dose administrated directly to the blood of an adult woman: 30 µg in ~5-liter blood, 0.06 µg/ml.
• “End-organ dose”: 1:1000 dilution of the “injected dose”. This concentration refers to the 0.1% accumulation of the administrated NLP vehicle at the ovaries of female rats as indicated in the “Final report”.

hpGCs were harvested 24 or 48 h later for RNA analysis and the culture medium was collected for protein analysis.

Sorry, I just have to question yet again the assumption that the entire vaccine dose distributes in the blood volume. OK, I’ll stop beating that dead horse.

Instead, I’ll beat this one. No change in cell viability was observed. The vaccine did not cause cell death:

The MTT assay measures cell viability by measuring how well the cells can convert a water-soluble substrate into an insoluble purple-colored formazan dye. Metabolically active cells can do it using enzymes called mitochondrial dehydrogenases; metabolically inactive (i.e., dead) cells cannot. MTT assays are commonly used—my lab has long used the assay—as a surrogate for how many viable cells are present in a dish. Although MTT assays are commonly used this way, they are subject to a number of drawbacks and confounders that can include cell seeding number, MTT concentration, MTT incubation time, serum starvation, cell culture media composition, released intracellular contents (cell lysate and secretome), and extrusion of formazan to the extracellular space. One key limitation is that MTT assay does not directly measure the number of viable, proliferating cells, but rather measures metabolism. I probably don’t need to harp on this too much, as there was no significant difference in any of the groups.

Next up, the authors looked at the mRNA levels of InhibinB and other selected genes after a 24-hour and 48-hour exposure to the vaccine:

Personally, I think the authors confused the issue by even including the “whole dose.” The “end-organ” dose is all that matters, and even then I question whether that dose isn’t massively too high. I also note that this is all mRNA. What really matters is protein levels. So let’s see:

Next, we measured the level of InhibinB protein secreted from the hpGCs into the culture medium, in response to vaccine exposure, and detected an increase in its level (112, 121, and 134% in three independent experiments, compared to control) after 48 h of exposure to the end-organ dose, matching the increase in its mRNA level. There was no detectible change in InhibinA protein level in the culture medium in either dose or exposure duration to the vaccine (data not shown).

Did you notice what I did? So apparently there was an increase in protein, but I find it very odd that they didn’t show the actual data in a graph form and only mentioned the protein levels after the 48 hr exposure to the “end-organ” dose. After all, look at the InhibinB mRNA level after 24 hours! That’s a nearly three-fold increase! Was there a three-fold increase in protein level at that point? Did the protein secreted into the media correlate for all time points? I rather suspect some cherry picking here. After all, they only mention the 48 hour exposure, and I really wonder whether a 12-34% increase in protein level is even biologically significant. I want to see protein levels for every exposure at every time point for every gene.

Oh, wait. In the discussion the authors admit:

Even though secretion of InhibinB into the culture medium was elevated compared to control in 3 independent experiments, it was not statistically significant.

Seriously, that should have been stated immediately. So basically there was no change in protein levels measured. I went and looked for a supplementary data section thinking that maybe they had included the data there. Nope. All that was there was a supplementary table listing the characteristics of the women undergoing IVF from whom hpGCs were harvested.

None of this stopped the authors from charging ahead with a study of five (!) women who reported menstrual changes after a third dose of vaccine:

Finally, we asked whether the detected increase in inhibinB, following a direct vaccine exposure, can also be tracked in vaccinated women and point to its role in menstrual irregularities. To that end, we analyzed blood samples from five women before and ~1 month after 3rd dose vaccination. All women reported changes in their menstrual bleeding pattern post the vaccine. Blood was collected without recording the day of the menstrual cycle. Since FSH/InhibinB present a relatively stable ratio, independent of the day of cycle²⁹, and as each woman has her own FSH/InhibinB ratio that is steady, not only throughout the cycle but in following cycles^30,31, we followed that ratio. We found that for all women tested, the FSH/InhibinB ratio was changed 2–3 folds post vaccination (Fig. 5).

OK, that sounds…suspicious. Oh, wait. They left something out, as Dr. Vicki Male noted:

This is where it gets a bit odd, because the authors say: "for all women tested, the FSH/InhibinB ratio was changed 2–3 folds post vaccination".

But they don't mention that in three of them the 2-3-fold change is up and in two of them it's down. 8/

— Viki Male (@VikiLovesFACS) July 2, 2024

Wait, what? While it’s true that the authors do show the data on a graph, it is rather bizarre not to state that the two- to three-fold changes observed could be two- to three-fold up or down! This is basically noise, likely random noise. Again, if I had been reviewing this paper, I would have insisted on seeing all the protein data in a supplementary section, and I would have called the authors out for not making it immediately clear that the InhibinB protein level changes reported were not statistically significant. In fact, I would have told them to remove that whole paragraph about the changes and simply add a sentence stating that there was no significant change in the level of protein secreted into the medium.

I would also have forced them to remove this paragraph from the Discussion section:

The levels of FSH and InhibinB change along the menstrual cycle, but their ratio remains relatively constant, independent of the day of cycle⁴³; this ratio is expected to be similar between consecutive menstrual cycles of the same woman. In line with our in-vitro experiments, our in-vivo analysis of the FSH/InhibinB ratio in women before and ~1 month after the 3rd dose of COVID-19 vaccine showed that the post vaccination FSH/InhibinB ratio was changes by 2–3 folds. This change reinforces out hypothesis that vaccination caused an immediate elevation in InhibinB expression, that led to changes in the menstrual cycles’ length and bleeding, as well as to an altered FSH/InhibinB ratio a month later.

No. It does not. I reinforces nothing; in fact, it goes completely against the hypothesis, given that the changes were basically random. How did this paragraph and the one above it in the Results section get past the reviewers?

Again, the lack of any actual result regarding COVID-19 vaccine and menstrual changes doesn’t stop the authors from speculating wildly:

As the anti-COVID-19 vaccine is the first commercially available mRNA-based vaccine, and since there is no available vehicle to serve as “control”, we cannot discard the possibility that the changes we characterized in the hpGCs were induced by the vaccine envelope and not specifically by COVID-19 mRNA sequence. Today, when there are more mRNA-based vaccines in the pipeline⁴⁴ this issue is highly relevant.

Maybe, but this study sure isn’t a good argument for its relevance. But do go on:

To summarize, this study reveals a unique, independent mechanism for vaccine related menstrual changes, concomitant with the vaccine-inflicted immune response. Our work suggests that at exposure, the COVID-19 vaccine can affect GCs directly, though not by reducing their viability. Exposure to the end-organ concentration of the vaccine exerted changes in the transcripts of two ovarian-regulatory key factors: a prominent upregulation of InhibinB and a downregulation of AMH. These changes can strongly affect FSH serum levels in vaccinated women; lead to disrupted follicular growth (i.e., too many follicles growth at the “wrong” time of the cycle) and activity (i.e., estrogen production); and ultimately affect the uterus cyclicity that is clinically displayed by changes in the menstrual bleeding pattern. Serum analysis of vaccinated women who reported menstrual changes, showed a transformed FSH/InhibinB level, supporting our results.

This study reveals no such “unique, independent mechanism for vaccine-related menstrual changes. It doesn’t even demonstrate the upregulation of the relevant protein in cell culture, much less in animals or humans. As important a scientific question as the mechanism behind the menstrual abnormalities observed after vaccination with the mRNA-based COVID-19 vaccines is, this study sheds almost no light on what that mechanism might be. It is mostly a negative study.

Worse, this study is scientific clickbait that managed to attract the attention of antivaxxers because it is scientific clickbait. NPG journals are definitely slipping here. This paper will provide an excellent negative case study for any faculty who want to teach how to critically review the literature.