In China, recent years have witnessed a continuous decrease in birth rate, adding burdens to the aging problem of the population. Many factors have led to this decrease, among which a prominent one is the increasing rate of male infertility. With growing pressure from various aspects, males of reproductive-age are suffering from the degeneration of sperm quality ^[1]. This year, a public survey held in Chongqing showed that 80% of the semen samples from undergraduates were unqualified. ^[2] The unsatisfactory sperm quality has become a consensus worry that requires public attention and prompt measures.

Actually, things are getting worse not only in China but globally. According to available data from the Centers for Disease Control in the U.S., 1 in 8 couples of reproductive-age experiences infertility, among which low sperm quality is considered as one of the major causes. ^[3] Another research revealed that the sperm quality of reproductive-aged men worldwide has dropped over 50% in the last 40 years. ^[4]

A deteriorating trend of sperm quality can be observed from studies over the last four decades in multiple Western countries. ^[5-6]

Considering the urgency of solving the problem of male infertility, as well as the hope of contributing to tackling the population aging issues in China, we came up with the idea of providing sperm quality detection product for household use.

As mentioned above, the urgency of promoting reproductive health has become increasingly prominent. To improve human reproductive health through assisted reproductive technology and semen screening, we went through literature research and human practice to find that both hospital and non-hospital solutions targeting such a significant issue were not well-developed at present.

In communication with a medical professor Dr. Xie Lan, we learned that current hospital examinations mainly focus on indicators like sperm concentration and motility by means of manual microscopic observation. Plenty of manpower and strenuous effort can be spared if these vital indexes of sperm quality could be examined with a rapid detection device. Aside from hospital examinations, there are some related sperm quality detection products on the market provided by vendors. However, no mature industry has been established given that these already existed household test strips remain in a relatively primitive stage of development, lacking accuracy and reliabilty.

Given the current development of sperm quality examination devices, it occurred to us that we could transform the standard sperm examination index into a more visualized signal that can be easily measured. Therefore, we came up with the idea of introducing synthetic biology and chip-based strips into household diagnosis of sperm quality.

At first, we got our inspiration from the pregnancy test kit. Developed in late 20th century, it took advantage of the highly specific antigen-antibody hybridization reaction to test the concentration of human chorionic gonadotropin (HCG) in the urine, exhibiting extraordinary sensitivity and short waiting time.

Inspired by pregnancy test kit, we intended to design a test strip capable of detecting vital indicators of sperm quality. One difficulty lies in the cultivation of engineered bacteria, which cannot be cultured in a simple paper strip. Another difficulty lies in the representation and integration of different signals indicating major indexs of sperm quality. To overcome these difficulties, we applied microfluidic chip consisting of microchannels with chemokine gradients to induce viable sperms to enter the detection area and integrate different signals detected through biomarkers on the sperm surface.

After extensive literature research, we narrowed our research direction down to the detection of semen vitality and determined two important characteristics reflecting sperm quality-----mobility and fertility. Sp10, also known as Acrv1, is the most well-known protein on the surface of mammal sperms (more accurately the acrosomes). Moreover, EGFR is another sperm surface protein reported to be involved in the fertilization between sperms and eggs. We selected Sp10 as the biomarker representing motility, while EGFR as the biomarker indicating fertility.

Subsequently, we were confronted with the obstacle of engineering the E. coli to recognize, relay, and represent targeted protein signals. Through literature research of the natural chemotaxis of prokaryotic cells, we came across the two-component system (TCS), which can respond to a variety of environmental stimuli. TCS is comprised of two components, a sensor kinase (SK) which receives stimuli and gets autophosphorylated, and a response regulator (RR) which in turn is activated and has a DNA binding domain to regulate gene expression. The TCS has two advantages. One is its speed as a result of simple signal transduction; the other is its high modifiability. We can modify the receptor of TCS to recognize the protein in interest.

To repond to two different protein signals, we engineered two TCSs to detect them respectively. On the one hand, we altered the ligand-binding domain of PmrB (sensor kinase of TCS PmrB/A) with an ntibody-binding domaina from an affibody. Prediction of AlphaFold and pre-experiments confirmed that our modification had no effect on the normal function of PmrB. In this way, we could use an antibody of sp10 to bridge the signal transduction between sp10 and the engineered PmrB/A TCS.

On the other hand, we fortunately found another type of affibody possessing high affinity to EGFR, which completely meets our demand. As a result, we fused this affibody with nisin, a modified peptide that can be recognized by NisK (sensor kinase of TCS NisK/R). Here, the fusion protein affi-nisin served as the mediator. So far, we accomplished our aim of surface protein detection and signal transduction by engineering sensor kinases of TCSs.

Another obstacle we need to deal with is that the two indexes we chose are actually correlated in a way that high motility is the premise of fertility. Therefore, not orthogonal but conditionally-associated circuits should be built. Provided that, we established a three-state logic gate with serine integrase Bxb1 and cro/cI multiregulation system

To be brief, a promoter is flanked by coding sequences of two color proteins and is shut off in the resting state by constitutively expressing cI. When the 4 (the mobility index) signal is transducted, cro is transcibed and derepresses the inhibition of cI, resulting in expression of color proteins. Meanwhile, the direction of the promoter is controlled by the existence of serine integrase Bxb1. When the EGFR (the fertility index) signal is transducted, Bxb1 is transcibed and inverts the direction of the promoter. It will be easier to comprehend our design using this truth table:

For more details on our wetlab design, please visit design page.

As for the hardware, we intended to design a microfluidic chip to test sperm quality. We allow affi-nisin and anti-sp10 antibody to premix with semen sample in the sample loading well. Then, the sperms would undergo capacitation and enter the detection well under the induction of chemokine gradients. Finally, visualizable results of sperm motility and fertility would be presented according to protein detection.

For more details on our hardware design, please visit hardware page.

The iGEM competition is just the start of our product. Here we present our future perspectives both on our project and the trend of public awareness of the reproductive concern.

Firstly, though applying synthetic biology in diagnosis and biosensors are not groundbreaking, we have foreseen the potentials of our protein-detecting system beyond the design in this project. In this system, we could simply modify the antibody fused with TCS to detect any protein in interest. What’s more, serine integrase and cro/cI system are capable of realizing more complicated logic gates since the three basic gates can be successfully constructed. In summary, we accomplished a modifiable logic operation system capable of processing protein signals. Apart from the application in sperm quality test demonstrated here，it can be used in scenarios like cancer biomarker identification, microscale protein detection, bacteria memory and computation, quick pathogen test and etc. With several parts modified, it will serve in diverse application scenarios. We are looking forward to exploring more interesting and meaningful applications of our system.

Secondly, we are offering a novel solution to the vacancy of household sperm quality test. The household access to self-examination will greatly facilitate relevant people. For the medical workers, it will free them of cumbersome manual microscopic observation burden. For the people who feel embarrassed or are inconvenient to pay hospital visits, household test will be more acceptable and less troublesome.

Lastly, we are contributing our effort to tackling reproductive concerns. Just as we have emphasized above, more public awareness should be raised. We are hoping that our project and human practices are calling for a positive attitude towards related problems and a promising future with less disharmony and discrimination. Each person, regardless of gender, race and status, will share the right of using reproductive household self-test and confront reproductive issues in open air. For more details on our future perspective, please visit implementation page.

[1] Liu, J., Dai, Y., Li, Y., Yuan, E., Wang, Q., Wang, X., & Guan, Y. (2020). A longitudinal study of semen quality among Chinese sperm donor candidates during the past 11 years. Scientific reports, 10(1), 10771.
[2]https://m.weibo.cn/status/4786179699900864?sourceType=weixin&from=10C9595060&wm=9006_2001&featurecode=newtitle
[3]https://storkotc.com/male-factor-infertility/
[4] Levine, H., Jørgensen, N., Martino-Andrade, A., Mendiola, J., Weksler-Derri, D., Mindlis, I., Pinotti, R., & Swan, S. H. (2017). Temporal trends in sperm count: a systematic review and meta-regression analysis. Human reproduction update, 23(6), 646-659.
[5]Auger, J., Eustache, F., Chevrier, C., & Jégou, B. (2022). Spatiotemporal trends in human semen quality. Nature reviews. Urology, 1-30. Advance online publication.
[6]Leaver R. B. (2016). Male infertility: an overview of causes and treatment options. British journal of nursing (Mark Allen Publishing), 25(18), S35-S40.