Human Reproductive System Research Articles

Human Female Reproductive System Organoids: Applications in Developmental Biology, Disease Modelling, and Drug Discovery.

Organoid technique has achieved significant progress in recent years, owing to the rapid development of the three-dimensional (3D) culture techniques in adult stem cells (ASCs) and pluripotent stem cells (PSCs) that are capable of self-renewal and induced differentiation. However, our understanding of human female reproductive system organoids is in its infancy. Recently, scientists have established self-organizing 3D organoids for human endometrium, fallopian tubes, oocyte, and trophoblasts by culturing stem cells with a cocktail of cytokines in a 3D scaffold. These organoids express multicellular biomarkers and show functional characteristics similar to those of their origin organs, which provide potential avenues to explore reproductive system development, disease modelling, and patient-specific therapy. Nevertheless, advanced culture methods, such as co-culture system, 3D bioprinting and organoid-on-a-chip technology, remain to be explored, and more efforts should be made for further elucidation of cell-cell crosstalk. This review describes the development and applications of human female reproductive system organoids. Graphical abstract Figure: Applications in developmental biology, disease modelling, and drug discovery of human female reproductive system organoids. ASCs: adult stem cells; PSCs: pluripotent stem cells.

Curcumin as an indirect methylation inhibitor modulates the effects of Toxoplasma gondii on genes involved in male fertility.

Toxoplasma gondii is a common protozoan parasite, which infects warm-blooded mammals, including mice and humans, throughout the world. The negative effects of T. gondii infection on the human reproductive system have been documented, especially in females. However, only few studies have examined the effects of T. gondii infection on the male reproductive system. Previous research shows that T. gondii can induce DNA methylation in some gene promoters, which are key regulators of spermatogenesis. Therefore, this study aimed to evaluate the effects of curcumin on the activity of DNA methyltransferases (DNMTs), as well as selected genes, involved in spermatogenesis in spermatogenic cells. In the spermatogenic cells exposed to T. gondii, there was a significant increase in DNMT1 and DNMT3A gene expression and a significant reduction in HSPA1A, MTHR, and DAZL gene expression, compared to the controls. The present results showed that curcumin could regulate changes in T. gondii-mediated gene expression. The effect of T. gondii on DNMT activity was also investigated in this study. A 40 % increase in DNMT activity was observed due to T. gondii infection. However, DNMT activity was restored by treatment with 20 μM curcumin for eight hours. The results revealed that T. gondii increases the NF-κB activity, compared to the control group. The increase in NF-κB activity, induced by T. gondii, was inhibited by curcumin. In conclusion, T. gondii, by increasing DNMT expression and activity, leads to an increase in NF-κB activity in cells. On the other hand, curcumin reduced DNA methylation, induced by T. gondii, owing to its NF-κB-inhibiting properties. Therefore, curcumin, as a hypomethylating agent, can be potentially used to alleviate the negative effects of T. gondii on the male reproductive system.

The Effects of E-Cigarette Vapor Components on the Morphology and Function of the Male and Female Reproductive Systems: A Systematic Review.

E-cigarettes, a comparatively new phenomenon, are regarded as a safer alternative to conventional cigarettes. They are increasingly popular among adolescents of both sexes, and many smokers use e-cigarettes in their attempts to quit smoking. There is little understanding of the effects of exposure to e-cigarette vapors on human reproductive health, human development, or the functioning of the organs of the male and female reproductive systems. Data on the effects of the exposure were derived mainly from animal studies, and they show that e-cigarettes can affect fertility. Here, we review recent studies on the effects of exposure to e-cigarettes on facets of morphology and function in the male and female reproductive organs. E-cigarettes, even those which are nicotine-free, contain many harmful substances, including endocrine disruptors, which disturb hormonal balance and morphology and the function of the reproductive organs. E-cigarettes cannot be considered a completely healthy alternative to smoking. As is true for smoking, deleterious effects on the human reproductive system from vaping are likely, from the limited evidence to date.

SARS-CoV-2 and the next generations: which impact on reproductive tissues?

Coronavirus disease 2019 (COVID-19) caused by the severe acute respiratory syndrome–coronavirus 2 (SARS-CoV-2) is a severe global pandemic, affecting mostly the respiratory system. Understandably, attention is also being directed towards the urogenital tract. In this work, expression patterns of various host molecules possibly involved in viral entry and replication were investigated in human female and male reproductive systems by inquiring online repositories, including the Human Protein Atlas, GTEx, FANTOM5. Our findings highlight that male reproductive tissues could be targeted by SARS-CoV-2, particularly the testis since it co-expresses the receptor (ACE2) and the protease (TMPRSS) needed for viral entry. We hypothesized that SARS-CoV-2 infection could have repercussions on the fertility status of male individuals Potential infectivity of SARS-CoV-2 in reproductive tissues should be considered in reproductive medicine and management of in vitro fertilization in present and future generations.Electronic supplementary materialThe online version of this article (10.1007/s10815-020-01917-0) contains supplementary material, which is available to authorized users.

Development of the Discovery With Team Assisted Individualization (D-TAI) Model to Empower Analytical Thinking Ability in the Material Human Reproductive System

The objectives of this research and development are: to find out the characteristics of the Discovery with Team Assisted Individualization (D-TAI) learning model, determine the feasibility of the D-TAI learning model, and test the effectiveness of the product model in empowering analytical thinking skills. Model development research refers to Borg & Gall, which was adapted into nine stages: research and data collection, planning, initial product development, initial testing, product revision I, main field revision, product revision II, operational field revision, and final product revision . Analysis of the data used is a qualitative and quantitative descriptive analysis using the Mann Whitney U test. The results of the research and development show: the development of the D-TAI model was carried out with regard to the characteristics of the model. the results of the development of the D-TAI model are applied to the material of the human reproductive system. D-TAI model is able to empower students' analytical thinking skills. The results of the study show a significant difference between the value of analytical thinking skills in the D-TAI model class and the control class that is the average value of the D-TAI model class (74.79) higher than the control class ( 55.91) control with sig level 0.00 <0.05.

New Sensitive Technique For Estimation Of Estrogen Receptors

Background: Estrogens play a key role in the human male reproductive system. Estrogen receptors (ERs: ERα and ERβ) was detected in germ cells from spermatogonia to spermatozoa.
 Objective: Tto evaluate the different estrogen receptors expressions on the sperm in infertile males using a new technique and apply that on infertile patient with varicocele.
 Materials and Methods: This study included 200 males, divided into two equal groups. Group1 included fertile males, and group II included infertile males tested according to presence of varicocele with different grades which were classified according to Dubin-Amelar clinical classification of varicocele (1970), 1 m pellet was examined for the expression of the ERα and ERβ using immunocytochemistry stain. The stained sperm was processed in Photoshop program. The estrogen receptors were visualized after magnification and increased resolution in addition using a yellow filter. Percentage of stained sperm with different location in relation to the unstained sperm was counted.
 Results: There was a strong expression of ER β on the sperm in fertile males while ERα expression very reduced in fertile males and almost absent in all infertile males. Also, there was a statistically significant decrease in both ERα and β expression in infertile males than fertile males and in varicocele.
 Conclusion: ER technical estimation using immunoassay was better than any other techniques assay because ER could be present, but not function due to dislocation and deposition into wrong position.

The Invisible Burden of the Vietnam War: From the Memoirs of a Soviet Officer

This article publishes and analyses an interview with Lieutenant Colonel V. V. Skoryak, a Soviet military specialist who took part in the Vietnam War for eleven months in 1970. The interview describes little-known facts about military advisers’ stay in the country, when they mostly stayed far away from the frontline and dealt with the preparation and maintenance of the S‑75 high-altitude air defence systems. Special attention is paid to the everyday life of the advisers and their legal status, which helps reveal new aspects of the “everyday history” of war. Skoryak speaks about the ideological, moral, and psychological preparedness of the Soviet people to fulfil their “international duty”, which, according to him, was internally motivated. He also analyses post-traumatic syndromes in Soviet military men: it was especially frequent and profound in the early stages of the conflict. Additionally, the interview contains information about the medical care provided to the participants of the conflict and the consequences for their health. It puts forward some ideas about how the chemical weapons used by the Americans affected the human reproductive system. The interview provides an emotional assessment of the war and their place in the biography of a Soviet officer.

Expression of RXFP2 receptor on human spermatozoa and the anti-apoptotic and antioxidant effects of insulin-like factor 3.

Insulin-like factor 3 (INSL3) has an important role in the human reproductive system; however, its detailed function is still mysterious. We aimed to investigate the possibility of expression of RXFP2 receptor on human spermatozoa and to determine the anti-apoptotic and antioxidant mechanism derived the binding of INSL3 and RXFP2. In this experimental study, the expression/location of the RXFP2 receptor was determined on the spermatozoa of fertile and infertile men. Twenty samples from 20 fertile men were collected and divided into 6 parts (control group, and five groups treated with INSL3 10, 100, 250, 500, 1,000ng/ml). DNA damage, active caspase, reactive oxygen species (ROS) and sperm parameters were evaluated by TUNEL, flow cytometry, optical microscope and computer-assisted sperm analysis. The expression of RXFP2 was confirmed by Western blot. Immunocytochemistry illustrated that this receptor is expressed in the posterior half of the spermatozoa's head. The INSL3 at concentrations of 500 and 1,000ng/ml reduced the active caspase and mitochondrial ROS, and also reduced DNA fragmentation at 1,000ng/ml. Besides, INSL3 500 and 1,000ng/ml significantly increased the sperm motility. This study confirmed the presence of RXFP2 receptor in fertile and infertile men's spermatozoa, indicating the highly dose-dependent efficacy of the INSL3, which may have promising impacts on the in-vitro fertilisation outcomes.

Recent Advances in Electrochemical Sensors and Biosensors for Detecting Bisphenol A.

In recent years, several studies have focused on environmental pollutants. Bisphenol A (BPA) is one prominent industrial raw material, and its extensive utilization and release into the environment constitute an environmental hazard. BPA is considered as to be an endocrine disruptor which mimics hormones, and has a direct relationship to the development and growth of animal and human reproductive systems. Moreover, intensive exposure to the compound is related to prostate and breast cancer, infertility, obesity, and diabetes. Hence, accurate and reliable determination techniques are crucial for preventing human exposure to BPA. Experts in the field have published general electrochemical procedures for detecting BPA. The present timely review critically evaluates diverse chemically modified electrodes using various substances that have been reported in numerous studies in the recent decade for use in electrochemical sensors and biosensors to detect BPA. Additionally, the essential contributions of these substances for the design of electrochemical sensors are presented. It has been predicted that chemically modified electrode-based sensing systems will be possible options for the monitoring of detrimental pollutants.

The toxicity effects and mechanisms of tris(1,3-dichloro-2-propyl) phosphate (TDCPP) and its ecological risk assessment for the protection of freshwater organisms

Tris (1,3-dichloro-2-propyl) phosphate (TDCPP) is a type halogenated organophosphate flame retardants (OPFRs), which has been identified as contaminants of emerging concern (CECs). The use and production of OPFRs began to increase gradually when brominated flame retardants (BFRs) were banned. Halogenated OPFRs, especially TDCPP have been considered to lead to mutagenicity and carcinogenesis and major concerns have been raised regarding their toxicity. In this study, the toxicity effects and mechanisms of TDCPP were summarized and ecological risk assessment was made regarding its potential impact on freshwater organisms. TDCPP has been widely detected in ecosystems throughout the world, with observed toxicity effects on both humans and freshwater organisms. Inhalation of the dust was found to be the main exposure for humans. TDCPP could be metabolized in the human body, and medium stability was achieved in human body with the main metabolite BDCPP. Aside from mutagenicity and carcinogenesis, TDCPP was also found to have the potential for endocrine disruption and impairing the human reproductive system. Furthermore, this study reviewed the results of previous toxicity experiments, including acute toxicity, growth and development toxicity, neurotoxicity, and hepatotoxicity in freshwater organisms. Risk assessment was made using the safety threshold method by comparing the toxicity data with the exposure data in freshwater. HC5 (hazardous concentration for 5% of organisms) derived based on traditional endpoints of acute toxicity LC50 (median lethal concentration) or EC50 (concentration for 50% of maximal effect) was 877 μg/L. This value was much higher than the exposure concentration levels in the surface water with EXD90 (exposure data with cumulative probability 90%) of 65.22 ng/L. However, based on the growth and development toxicity data, the derived HC5 was 33.33 ng/L and the calculated MOS (margin of safety) was below 1. Therefore, the results validated the fact that the ecological risk of TDCPP could not be neglected for its growth and development toxicity.

Lesson Study for Learning Community through Mind Mapping in Biology at Senior High School

The study aimed to describe the performance of the learning community (LC) involving teachers and students. In the learning, mind mapping (MM) was used in the material of the human reproductive system through the activities of Lesson Study (LS). The study was conducted at MAN 2 Kota Madiun, in the odd semester of the 2018/2019 academic year. The study was conducted for eight discourses. The performance of LC data was collected using the Professional Learning Community (PLC) sheet. The result showed that LC performance increase by the time of discourse implementation. At the end of the study, LC performance reached 84.78% and 97.82% for the lowest and highest values, respectively. This result interprets that LS activities lead LC with very good criteria. The conclusion of the research showed that LC through the use of LS-based mind mapping on the material of the human reproductive system was good.

Axisymmetric mixed convective propulsion of a non-Newtonian fluid through a ciliated tubule

Thousands of chemical reactions occur in the human body when certain biological fluids, such as blood, semen, mucus, and synovial joint materials, move in various organs. These reactions play a vital role in regulating the life sustaining metabolic processes in the body. Analysis of thermal effects on these chemical reactions is relatively a new area in modern clinical medications. The present study investigates a simulation of the combined response due to heat and mass transport mechanisms taking place in the human body during the flow of physiological fluids. In particular, we focus our attention on the human male reproductive system, wherein the semen transports through the ductus efferentes due to metachronal waves of cilia. The constitutive relations of the robust Jeffrey viscoelastic fluid are used to model the human semen. The mathematical model of the present problem constitutes the axisymmetric flow of a Jeffrey fluid inside a vertical tubule under the influence of mixed convective heat and mass transfers. The inner side of the tubule is covered with ciliated structures. The influence of thermal behaviors of various metabolic processes in the human body due to an external heat source or sink is also taken into account. The mathematical formulation consists of using the approach of lubrication theory approximation; the nonlinear momentum, energy, and concentration equations are simplified to get analytical solutions. Explicit expressions for temperature, concentration, velocity, pressure gradient, and volume flow rate of the proposed bodily fluid (i.e., human semen) are formulated. The expression for the volume flow rate is used to estimate the volume flux of the semen under the influence of various parameters. A comparison between the theoretical and experimentally obtained values of the flow rate of the human semen is also made. It is noted that our calculated values are very close to the estimated values. Industrial applications of the present results are obvious in the fabrication of artificial cilia pumping systems for microfluidic flow systems.

Single-cell transcriptome analysis of the novel coronavirus (SARS-CoV-2) associated gene ACE2 expression in normal and non-obstructive azoospermia (NOA) human male testes.

Being infected by SARS-CoV-2 may cause damage to multiple organs in patients, such as the lung, liver and heart. Angiotensin-converting enzyme 2 (ACE2), reported as a SARS-CoV-2 receptor, is also expressed in human male testes. This suggests a potential risk in human male reproductive system. However, the characteristics of ACE2-positive cells and the expression of other SARS-CoV-2 process-related genes are still worthy of further investigation. Here, we performed singlecell RNA seq (scRNA-seq) analysis on 853 male embryo primordial germ cells (PGCs) and 2,854 normal testis cells to assess the effects of the SARS-CoV-2 virus on the male reproductive system from embryonic stage to adulthood. We also collected and constructed the scRNA-seq library on 228 Sertoli cells from three non-obstructive azoospermia (NOA) patients to assess the effects at disease state. We found that ACE2 expressing cells existed in almost all testis cell types and Sertoli cells had highest expression level and positive cells ratio. Moreover, ACE2 was also expressed in human male PGCs. In adulthood, the level of ACE2 expression decreased with the increase of age. We also found that ACE2 positive cells had high expressions of stress response and immune activation-related genes. Interestingly, some potential SARS-CoV-2 process-related genes such as TMPRSS2, BSG, CTSL and CTSB had different expression patterns in the same cell type. Furthermore, ACE2 expression level in NOA donors’ Sertoli cells was significantly decreased. Our work would help to assess the risk of SARS-CoV-2 infection in the male reproductive system.Electronic Supplementary MaterialSupplementary material is available for this article at 10.1007/s11427-020-1705-0 and is accessible for authorized users.

Sample Treatment Methods for the Determination of Phenolic Environmental Estrogens in Foods and Drinking Water.

Environmental endocrine-disrupting chemicals (EDCs) are a large category of chemicals that can disrupt normal endocrine functions of organisms. They can enter the environment and foodstuffs in different ways and bring great threat to human health. Phenolic environmental estrogens (PEEs) are the typical representatives of EDCs and include bisphenols, alkylphenols, and others such as dibromophenol, dichlorophenol, 4-nitrophenononylphenol, etc. Even trace amounts of PEEs can cause serious damage to the human reproductive, nervous, and immune systems; therefore, it is very important to develop accurate and sensitive methods for their determination in different matrixes. At the present, there are still many challenges in the determination of PEEs in the samples with complex matrixes, and the sample treatment is one of the key issues. This review deals with the recent advances in sample treatment methods for the analysis of PEEs in foods and drinking water, including improved and newly developed liquid-liquid extraction, solid-phase extraction (SPE), magnetic SPE, matrix solid-phase dispersion with new adsorbent materials, and stir-bar sorptive extraction. In addition, the challenges and perspectives are also briefly discussed.

Claudins: New Players in Human Fertility and Reproductive System Cancers.

Claudins are major integral proteins of tight junctions (TJs), the apical cell–cell adhesions that enable maintaining polarity of epithelial cells, their differentiation, and cell signaling. A number of studies have indicated that claudins might play a crucial role in both physiology and pathogenesis. Their tissue-specific expression was originally linked to the development of different types of cancer and triggered a hope to use them as diagnostic or prognostic markers. However, it seems that their expression is more complex than that, and undoubtedly, claudins participate in one of the most important molecular events in cells. This review summarizes the recent research evaluating the role of claudins in fertility and the most common endocrine-dependent cancers in the reproductive system and highlights the crucial role of claudins both in human fertility and the most common cancers.

Human reproductive system microbiomes exhibited significantly different heterogeneity scaling with gut microbiome, but the intra‐system scaling is invariant

Maintaining sexual reproduction in a highly competitive world is still one of the major mysteries of biology given the apparently high efficiency of asexual reproduction. Co‐evolutionary theories such as the Red Queen hypothesis would suggest that the microbiomes in human reproductive systems, specifically the microbiomes contained in semen and vaginal fluids, should reach some level of homogeneity thanks to arguably the most conspicuous microbiome transmission between two sexes. The long‐term sexual coevolution should favor the dynamic homogeneity or stability, which should also be beneficial for sexual reproduction such as sperm survival or fertilization on physiological/ecological time scale. We present a piece of quantitative evidence in the form of microbial community spatial heterogeneity to support the stability notion by analyzing three big datasets of the human vaginal, semen and gut microbiome. Methodologically, we applied a recent community‐level extension to the classic Taylor's power law, which reached the rare status of ecological law and has found applications beyond biology. Both ecological and evolutionary theories, such as hologenome/holobiont and Red Queen, as well as consideration of first principles, would predict that microbiome transmissions between two sexes should have homogenizing effects on the composition and stability of the microbiomes in human reproductive systems, and therefore have similar variance structures. This is supported by the finding that the power law analysis revealed human vaginal and semen microbiomes exhibited the same scaling parameter size in their community spatial (inter‐individual) heterogeneities, while the both exhibited significantly different heterogeneity scaling parameter size with the human gut microbiome.

The implementation of constructivist modules to correct misconceptions on the human reproductive system concept

It is necessary to find appropriate learning solutions to solve students’ misconceptions on the human reproductive system concepts. This study aims to determine whether learning through the implementation of a constructivist module influences the reconstruction of students’ knowledge about the human reproductive system. The method used in this study was experimental with one group pre-test and post-test design. The population consisted of 272 students from science class XI of SMAN 10 Fajar Harapan Banda Aceh and SMAN Modal Bangsa Aceh Province. A total of 107 students were chosen by random sampling. The study was carried out over four months from March to July 2019. The supporting instruments used were the learning media and the data collection instruments in the form of multiple-choice test questions with Certainty of Response Index (CRI). The data collected through a pre-and post-test were analyzed using percentages. The results show that the number of students who understand certain concepts rose by 52.36%, the number of students who had misunderstandings decreased by 43.58%, and the number of students who had misconceptions fell by 9.94%. So, it is concluded that learning through the constructivist module is effective in improving concept reconstruction and students’ learning outcomes.

Human reproductive system module based on Qur’an and hadith to improve students’ motivation and learning outcomes

This research was carried out on the development of learning modules about the human reproductive system based on the Quran and hadiths in West Labuhanhaji at a junior high school to improve students’ motivation and learning outcomes. The research and development method (R&D) with a Borg and Gall model was used in this study. The research population was students of class IX at in West Labuhanhaji, by a total of 92 students. A sample of 48 people was obtained through a purposive sampling technique. Data collection used test questions and motivation questionnaires. Data analysis was performed by using a t-test, with the acquisition of tcount > ttable at a significance level of 0.05 at 6.414>1.71, which means that there were significant differences in learning outcomes between the experimental and the control class. Learning motivation was assessed by using the self-determination method resulting in an average value of 4.69, meaning it is in a very good category. The results of the correlation between motivation and learning outcomes showed the value of tcount > ttable which was 4.227> 1.71 with the value of r approached +1 was 0.667 which means there was a positive correlation between motivation and learning outcomes.

Reproductive Health Status of Indian Women: A Critical Appraisal

Reproductive health is fundamental to social and economic development of a family, community as well as nation, and a key component of an equitable society. Reproductive health is important for gender equality and women empowerment. Women’s reproductive health status is poor, and their sexual and reproductive rights are not fully raised in many countries, maternal mortality rates are higher, and women’s, chances of dying of pregnancy-related complications are almost 50 times higher in developing countries than in developed countries. Women are particularly vulnerable and also have a lack of knowledge regarding reproductive health in India. Reproductive health is a concept of human rights. Important areas of concern for reproductive health programmes in India are poor quality of reproductive health services especially in an urgent situation; lack of focus of adolescent’s knowledge on reproductive health and lack of education. Women in India and particularly the economically disadvantaged women suffer the highest rates of complications due to pregnancy such as sexually transmitted diseases, and reproductive cancers. Lack of access to comprehensive reproductive care is the main reason, and many women suffer and die. Women are deprived of access to reproductive health care services and are influenced by the socio-economic cultural factors. Which include low social status in family and community, lack of access to economic resource and education, inability to make a decision about their health, nutrition and so on? Reproductive health facilities at the community level are poorly equipped to deal with gynecological and obstetric. Reproductive health is defined as a ‘state of complete physical, mental and social well-being and not merely the absences of disease or infirmity, in all matters relating to the reproductive systems and to its functions and processes’ (United States: 1994). Reproductive health addresses the human sexuality and reproductive processes, functions and system at all stages of life and implies that people are able to have a satisfying and safe sex life and that they have the capacity to reproduce and the freedom to decide, when and how often to do so. The main objectives of this study are; to study the reproductive health situation in an Indian context, to know the problems of women in respective of their reproductive system, the study aims at assessing the reproductive health status of Indian women, and to understand the relationship between reproductive health and gender rights.

The Influence of Thinking Aloud Pair Problem Solving (TAPPS) Learning Model Charged Science Literacy on Students Learning Competencies in the Material of Human Reproductive System at SMAN 1 Enam Lingkung

The Problem of the research at SMAN 1 Enam Lingkung was that the Biology learning process is less varied and teacher-centered, students were unable to solve problems in learning, low learning competencies of students from aspects of knowledge, attitudes, and skills,and low science literacy abilities of students. The purpose of this study was to determine the effect of applying thinking aloud pair problem solving learning model with science literacy on students learning competencies in human reproductive system material at SMAN 1 Enam Lingkung.This type of research is an experiment with a randomized control group post-only only design. The population was all class XI of SMAN 1 Enam Lingkung which were registered in the 2018/2019.Sampling was done using cluster random sampling, and the chosen sample was the XI MIPA 2 class as the experimental class, and the XI MIPA 1 class as the control class. The instruments used were in the form of posttest questions for knowledge competency, observation sheets for attitude and skill competencies. Based on the results of the knowledge competency research, the tcount was 5.14> ttable 1.67, the competency attitude was tcount of 15.12> ttable 1.67, and in the competency of skills tcount was 9.45> t table 1.67. This is an aceppted hypothesis. It was concluded that the model thinking aloud pair problems solving with sains literacy improve the competencies of knewledge, attitude, and skills in the material of the human reproductive system at SMAN 1 Enam Lingkung.