The people behind the papers – Manoj Madhavan and Ripla Arora

Manoj Madhavan (L) and Ripla Arora (R)

Ripla, can you give us your scientific biography and the questions your lab is trying to answer?

RA: I received my bachelor's and master's degree in Biochemistry from the University of Delhi in India. As part of my master's programme I took a course in developmental biology. I was completely fascinated by the beauty of the embryo's developmental programme and how this process occurs repeatedly with the same precision, embryo after embryo. This led me to pursue a PhD in genetics and development at Columbia University, New York, where I studied the role of T-box transcription factors in vascular morphogenesis of the allantois (umbilical precursor) and epithelial branching in the lung. I then completed my postdoctoral work at the University of California, San Francisco, where initially I worked on sex differentiation of the bipotential gonad and studied spatial aspects of the embryonic ovary. Motivated by the importance of spatial analysis in tissues, I went on to develop a novel technique for 3D imaging and quantitative modelling of the mouse uterus during preimplantation stages. As I was developing this method, I realised that, although much is known about the molecular maternal-foetal interactions during implantation, the 3D architecture of the uterine environment in which the early embryo develops is not well understood. Thus, I started my own research group at the Department of Obstetrics, Gynecology and Reproductive Biology, and the Institute for Quantitative Health Science and Engineering at Michigan State University in 2017. My team uses confocal imaging, in combination with 3D shape analysis, to identify and quantify dynamic changes in the luminal and glandular structure of mouse uterus in preparation for implantation. Further, we analyse the uterine shape in genetic mutants or hormonal perturbations that cause implantation defects, to uncover novel molecular pathways and global structural changes that contribute to successful embryo implantation. Our studies have implications for understanding how structure-based embryo-uterine communication is key to determining an optimal implantation site, which is necessary for the success of a pregnancy.

Manoj, how did you come to work with Ripla and what drives your research today?

MM: When I applied for the graduate program at Michigan State University, I was interviewed by Ripla. She seemed very amiable and helpful. Later, when I joined the program, I did a rotation in the Arora lab. I was intrigued by the lab's research on embryo-uterine interactions during early pregnancy. I was particularly fascinated by the new 3D imaging and 3D reconstruction methodology that Ripla had developed. Moreover, I was inspired by Ripla's passion and enthusiasm for her research. Hence, I decided to work with Ripla for my graduate studies. I believe I have an opportunity to use a powerful technique to uncover novel and exciting information on the 3D uterine structure during pregnancy, which may not be possible with the traditional techniques used in the field. What drives my research today is the hope that my data can be translated to the clinic to develop new approaches for diagnosis and treatment of infertility, and in assisted reproductive technologies.

Can you give us the key results of the paper in a paragraph?

RA & MM: We show that the uterus in wild-type mice exhibits dynamic changes in the uterine luminal folding pattern during the pre-implantation period. It forms transverse folds a few hours prior to implantation that then resolve to form flat implantation regions. Embryos attach in the centre of the flat regions and form a V-shaped chamber at the site of attachment. Formation and elongation of this V-shaped chamber enables rotation of the blastocyst-staged embryo to align the axis of the embryo with the uterine axis. Mutants with an aberrant folding pattern display longitudinal folds prior to implantation. Unlike the transverse folds, these longitudinal folds are unable to resolve at implantation regions and the embryos are trapped in these folds. Embryo trapping leads to disruption in chamber formation, followed by failure of embryo-uterine axes alignment, ultimately leading to defective embryo morphogenesis and embryo lethality later in pregnancy.

What is the function of the longitudinal folds at the earlier time points?

RA & MM: Longitudinal folds are observed when the embryos first enter the uterus. We have previously shown that clusters of embryos enter the uterine tube, move unidirectionally towards the middle of the uterine horn along the ovary-cervix axis. These clusters then scatter and space out bi-directionally along the length of the uterus. We predict that the longitudinal folds during the earlier time points serve as conduits for the unidirectional movement of embryo clusters from the oviductal end towards the middle of the horn.

Do you think that transverse folds are formed passively, to resolve the longitudinal folds, or do they play an important functional role?

RA & MM: Our current data suggest that formation of transverse folds coincides with resolution of longitudinal folds. Since there is a drastic shape change involved, we believe this is an active process requiring coordinated molecular signalling. However, our current data do suggest that transverse folds themselves may not have a functional role. In support of this idea, although the Wnt5a and Rbpj mutants that we used in our study have predominantly longitudinal folds, embryos that escape these longitudinal folds are still able to implant correctly and make it to birth. To conclusively discern if transverse folds have a functional role, we would need a fold-less mouse model, which is of interest to us for our future studies.

Is it possible to manipulate the size of the flat peri-implantation region and do you think this would have any consequences for implantation or pregnancy outcomes?

RA & MM: We observe that the length of flat peri-implantation regions depends on the number of embryos in the horn and the spacing between the embryos. Horns that have fewer embryos have longer peri-implantation regions and horns that have more embryos have shorter peri-implantation regions. Since peri-implantation regions form before embryos arrive at potential implantation sites, it is highly possible that the formation of peri-implantation regions dictates the spacing among embryos. Hence, we propose that the size of the flat peri-implantation regions may have an impact on embryo spacing but not on implantation or pregnancy.

When doing the research, did you have any particular result or eureka moment that has stuck with you?

MM: When we observed longitudinal folds instead of transverse folds in the Wnt5a and Rbpj mutant mice, we predicted that embryos would be trapped in the longitudinal folds during implantation. Indeed, we did find that embryos were trapped in longitudinal folds. But what was really surprising and exciting was that almost all the embryos trapped in longitudinal folds had their embryonic axis misaligned with the uterine axis. This was a key discovery as it was known that a misaligned embryonic axis by itself can result in pregnancy failure, and thus we were able to connect uterine structural defects (longitudinal folds) with poor pregnancy outcomes (loss of embryos at mid-gestation). This has now opened new doors for examining how uterine structure-based mechanisms facilitate embryo-uterine axes alignment.

And what about the flipside: any moments of frustration or despair?

MM: We first discovered the aberrant folding phenotypes in Wnt5a mutant mice and we worked primarily with this model to study the role of uterine folding. To definitively show that the pregnancy phenotypes we observed in the Wnt5a mutants were due to the aberrant folding pattern and not due to other effects from the loss of Wnt5a signalling, we searched for another mouse model with aberrant folds. We needed to confirm that aberrant folding in any mouse model would result in similar effects on embryo-uterine axes alignment and in poor pregnancy outcomes. We examined several different genetic mutant mouse models and tried many pharmacological treatments to disrupt folding, but we were mostly unsuccessful. Finally, we came across the Rbpj mutant mice that were known to have embryo-axis alignment defects. We predicted that they would have longitudinal folds and thus would phenocopy the Wnt5a mutant mice, and that is exactly what we found.

What next for you after this paper?

MM: As I mentioned earlier, I would like to apply my findings to more translational aspects, in addition to understanding basic biological mechanisms. Currently, I am studying how excess hormone levels as a result of hyperstimulation during in vitro fertilization alters the uterine structure, specifically the folding pattern, leading to compromised pregnancy.

Where will this story take your lab next?

RA: In our attempts to solve the puzzle of the role of embryo-uterine interactions in successful implantation, we have uncovered two pieces so far: precise embryo movement patterns (Flores et al., 2020) and uterine luminal folding (Madhavan et al., 2022) during the peri-implantation period. The next piece of this puzzle is examining the contribution of uterine muscle contractions in coordinating the movement of the embryos in the dynamically folding uterine luminal epithelium. We believe that doing so will help us build accurate models of key processes that modulate embryo movement. Another direction the lab is invested in is understanding how ovarian hormones – oestrogen and progesterone – regulate embryo movement, uterine folding and muscle contractions to facilitate even embryo spacing and implantation site formation. We believe that understanding these different aspects will help us in developing methods to regulate embryo movement for embryo attachment to the uterine tube, which is key for improving implantation success in the clinic when using artificial reproductive technologies.

Finally, let's move outside the lab – what do you like to do in your spare time?

RA: I enjoy painting and have recently discovered the chaos and pure joy of creating art using poured paint. I also enjoy yoga, dancing and cooking lamb curry.

MM: I enjoy cooking, going on walks and exercising. I also like to listen to music and catch up on my favourite TV shows.