Study that Impregnated Male Rats Stirs Controversy

A combination of approaches, including uterus transplantation and the joining of two animals’ circulatory systems, allowed males to bear pups, according to a preprint. But some experts say the experiments were not justified.  

Andy Tay
Jun 25, 2021
An illustration of the experimental setup, including the surgical joining of a male and female rat, the transplantation of a uterus into the male, and the transfer of embryos into both animals’ uteruses
Modified from © iStock.com, Hein Nouwens; The Scientist Staff

When researchers castrated a male rat, implanted a uterus into the animal, surgically joined its circulation to that of a female rat, and transferred embryos into the uteruses of each animal, they found that the male could in fact carry a pregnancy. In 4 percent of cases, pups that were carried by male rats and delivered through Cesarean section survived. 

The authors of the study, posted as a preprint on bioRxiv on June 10, say that this model could serve as a useful way to study reproductive biology, including identifying key factors in blood that could help maintain pregnancy. But some researchers question the utility of experiments using these highly artificial conditions, and the authors got so much pushback from the scientific community and the general public that they at one point requested the study’s retraction from the preprint server. For now, the preprint remains available.

“While the surgical manipulations necessary to generate the parabiotic animals used in these experiments are very delicate and must have required great skill, I question the authors’ assertion that they have created a model of male pregnancy with any real relevance for the study of reproduction,” Tony Wilson, a biologist at Brooklyn College who studies the evolution of reproduction, tells The Scientist in an email. 

As a graduate student at the Naval Medical University in Shanghai, China, in 2015, Rongjia Zhang read about the use of so-called parabiosis, the joining of two animals’ circulatory systems, to study aging and regeneration in rodent models. She began to wonder about the possibility of using this technique to provide a male rat with the hormones and other biomolecules associated with pregnancy, possibly allowing it to gestate a fetus. 

“I had the idea of using a model of [a] heterosexual parabiotic pair consisting of a castrated male rat and a female rat to study male mammalian pregnancy, but doubted the feasibility of this idea at first as it had not been done before,” Zhang tells The Scientist. “I spent a lot of time thinking, reviewing the literature, doing preliminary experiments, repeatedly modifying my plan before finalizing the experiments with Professor Liu Yuhuan, an experienced obstetrics and gynecology doctor who supervised me the last six years.” 

The final experimental plan consisted of four main steps. First, Zhang and her colleagues castrated male rats and surgically connected them to females along their sides, creating parabiotic pairs. For each pair, they then transplanted a uterus into the male before implanting blastocyst-stage embryos into both that uterus and the native uterus of the female. Finally, two days before the pregnancies would be considered full term, the researchers performed Cesarean sections and separated the adult rats. 

This study certainly gives one much to ponder from the philosophical to the logistical.

—Clint Kelly, Université du Québec à Montréal

The team found that in just more than half of 46 parabiotic pairs, neither the male nor the female became pregnant with normally developing embryos. In about one-third of the pairs, only the female ended up gestating normal embryos. And in just six (13 percent) of the pairs, both the male and female rats hosted normally developing embryos. No pair had normal embryo development in only the males, suggesting that the transplanted embryos could develop normally in the transplanted uteruses of male parabionts only when the female counterparts were pregnant and could provide the necessary blood-borne factors. 

“I doubt this research will soon translate into an epidemic of male mammals giving birth but I can envision the techniques and knowledge developed by such research advancing female reproductive health as well as other areas of medicine like organ transplant,” Clint Kelly, a biologist at the Université du Québec à Montréal who studies studies sexual selection and evolutionary biology, tells The Scientist in an email.

Others disagree. Paul Robbins, a biologist at the University of Minnesota, says that the stress of multiple surgeries and wound healing that followed introduced confounding factors that make it impossible to conclude that the pregnant blood microenvironment was the key to a successful male pregnancy. Certainly, blood from the pregnant female is not the only factor, adds Reza Ardehali, a cardiologist at the University of California, Los Angeles medical school, who has published a mouse parabiosis protocol. “The authors showed that the male parabiont had to be castrated in order to suppress male hormones,” says Ardehali. 

Most of the fetuses removed from the parabionts died within hours of delivery, which the authors attribute to early termination of the pregnancies. About 50 percent of the fetuses from the female parabionts died within two hours, while 67 percent of the fetuses from male parabionts did. Many of the dead fetuses from male parabionts had unusual characteristics such as darker skin morphology and degenerated or swollen placentas. In total, only 4 percent of embryos transplanted into the uteruses of male parabionts survived.

The results show that male pregnancy in mammals is possible, says Robbins. Ardehali agrees. “On a theoretical basis, this research strikes me as first-order plausible. Theoretically, what they describe could be accomplished, but practically, after the peer review, other groups must be able to replicate it.”

Wilson notes that with the extensive manipulations required, the authors have “effectively replaced the male reproductive system with that of a female, creating something that cannot reasonably be called male pregnancy.” 

“This study certainly gives one much to ponder from the philosophical to the logistical,” says Kelly. “But one obvious conclusion is that females are not simply males with a uterus, as any biologist could have told you prior to this study being conducted. Successful pregnancy clearly requires more than a uterus and sufficient exposure to progesterone and estradiol.”

From online criticisms to a requested retraction

The preprint has been widely shared and discussed online (as of June 23, it had been tweeted more than 3,000 times), with some readers speculating that the study supports the idea that pregnancy could now be possible in male humans—something Zhang says her study cannot speak to. Within a day of posting her study to bioRxiv, Zhang submitted a revision urging readers to be more cautious in their interpretations of the paper. 

“Our research started purely as a scientific curiosity, and we did not expect there would be so many nonscientific feedbacks on our experiments when our preprint was posted,” Zhang tells The Scientist. “We had to remind readers that the preprint is a work in progress and to correct their misconceptions.” 

But even among the scientific community, a number of researchers have voiced concerns about the experiments. Wilson writes: “Simply asserting that these results ‘may have a profound impact on reproductive biology’ is insufficient. Without this broader justification, the paper is essentially a technical exercise, motivated by the engineering credo, ‘Can we do this?’, rather than any more substantive attempt at biological understanding.”

Zhang emphasizes that her research was conducted under the strict supervision of a bioethics committee, and amid the criticism, she and her coauthor reached out to bioRxiv to retract the study. However, they then emailed bioRxiv again to stop the retraction, the preprint server confirms to The Scientist. “We have sent a letter to Biorxiv to stop the retraction,” Zhang writes in a recent post on PubPeer. “We don’t know whether our retraction will be stop, but to be honest, we did nothing wrong, just performed an animal experiment.” 

Zhang says she feels she is a victim of online bullying as a result of the study, though she declined to provide specifics on this point as her work has not been published and she does not want her comments to jeopardize the peer review process. “We have spent many years on this paper, and all the work we have performed is out of personal curiosity,” she tells The Scientist. “It is inappropriate to equate the conclusions of the preprint as scientific facts and exaggerate the implications.”

This study obviously has profound social ramifications, as well as posing serious questions of justification/necessity.

—Owen Schaefer, National University of Singapore

Arthur Caplan, a bioethicist at New York University School of Medicine, says he is not surprised that an animal review committee approved this study. “When reviewing studies, the animal review committee looks at whether the animals suffer from pain and how the pain is mitigated, and not the implications or future applications. It’s true that in this study, the parabiotic model might cause some pain, but it’s likely not an important [enough] factor to block the study.”

Owen Schaefer, a bioethicist at the National University of Singapore, tells The Scientist in an email that “the description of ethics approval [in this study] is indeed very thin and at the very least, it should be clarified which animal welfare bodies reviewed and approved the study, for the sake of accountability. Although for most studies, the description of ethics approval is thin, a good case can be made here as this study obviously has profound social ramifications, as well as posing serious questions of justification/necessity.”

While Zhang says it was not her intention to explore the possibility of male pregnancy in humans, bioethicists who spoke with The Scientist say that the authors should have been aware that some would interpret their study as a first attempt at just that. Caplan says that scientists may feel the need to hype the implications of their research to get more funding, without considering that the press may pick up their projects and could put a sensationalistic spin on them. Indeed, multiple news agencies from China to the United Kingdom have reported about this study.

“[Researchers] could try to be respectful of public misunderstandings and fear, and find ways to resolve them,” Caplan notes. “In Zhang’s case, if she had not included the last sentence in her preprint”—which reads, “Our experiment reveals the possibility of normal embryonic development in male mammalian animals, and it may have a profound impact on reproductive biology.”—“the preprint might not have gotten so many unrelenting criticisms and emotional critiques online.”

Schaefer adds that the authors only included a brief and vague sentence about the implications of their study, but the use of animals in a project that “fundamentally reorganizes biological functioning needs strong justification. The elephant in the room is that the reason this research is potentially impactful is that it could eventually be developed for human reproduction, i.e., for male humans to be able to become pregnant. It is hugely controversial, though, whether it’s even desirable to develop the possibility for male humans to reproduce.”

Caplan adds that this current research in reproductive biology will not be the only one to receive significant media attention. “I am aware of research in Japan on artificial wombs and artificial amniotic fluid, and the scientists working on these projects are not prepared to engage society in bioethics conversations.” One way to improve the current situation, Caplan says, is to introduce bioethics training for researchers that covers social responsibilities of science.

Schaefer also suggests that while it is too late to stop this study, a moratorium could be considered on similar future research, much like the moratorium on human germline editing that was proposed after the birth of the first gene-edited human babies. “Indeed, it won’t surprise me if pretty soon in light of this study and other developments, we see statutory bans on human applications of inducing male pregnancy being passed.”

Human applications aside, Wilson reiterates that the study lacks a defensible relevance. “While I am not a scientific ethicist, and recognize the value of animal models for the study of biological phenomena of both academic and clinical interest, the authors have to my mind failed to provide a clear and compelling justification for their experiment and how the generation of a mammalian model of male pregnancy could help to generate insights of broader significance.”