Human Reproductive Development Research Articles

Investigating the role of phenylalanine residues for amyloid formation of the neuropeptide neurokinin B

Neurokinin B (NKB) is a tachykinin peptide that has diverse roles in biology, including in human reproductive development. Cellular processing of this peptide is thought to involve formation of a dense core vesicle during transit through the regulated secretory pathway. The ability of NKB to rapidly form an amyloid can contribute to formation of the secretory granule but features that support amyloid formation of NKB are not well understood. NKB contains a diphenylalanine sequence well recognised as an important motif for self-assembly of other peptides including amyloid β. Using mutations of the diphenylalanine motif we show that this motif in NKB is necessary for amyloid formation, and it is the unique combination of aromaticity and hydrophobicity of phenylalanine that is crucial for aggregation. Using disulfide cross-linking we propose that phenylalanine at sequence position 6 is important for stabilising inter-sheet interactions in the NKB amyloid fibril. Although having a highly conserved sequence, the NKB peptide from zebrafish only contains a single phenylalanine and does not fibrillise as extensively as mammalian NKB. Analysis of self-assembly of NKB-like peptides from different species may help in elucidating their biological roles. Taken together, this work shows that mammalian NKB has evolved, within only 10 residues, a sequence optimised for rapid self-assembly, whilst also containing residues for metal-binding, receptor binding and receptor discrimination.

Replication stress in mammalian embryo development, differentiation, and reprogramming.

Duplicating a genome of 3 billion nucleotides is challenged by a variety of obstacles that can cause replication stress and affect the integrity of the genome. Recent studies show that replication fork slowing and stalling is prevalent in early mammalian development, resulting in genome instability and aneuploidy, and constituting a barrier to development in human reproduction. Genome instability resulting from DNA replication stress is a barrier to the cloning of animals and to the reprogramming of differentiated cells to induced pluripotent stem cells, as well as a barrier to cell transformation. Remarkably, the regions most impacted by replication stress are shared in these different cellular contexts, affecting long genes and flanking intergenic areas. In this review we integrate our knowledge of DNA replication stress in mammalian embryos, in programming, and in reprogramming, and we discuss a potential role for fragile sites in sensing replication stress and restricting cell cycle progression in health and disease.

Artificial intelligence algorithms for optimizing assisted reproductive technology programs: A systematic review

Artificial intelligence (AI) has been experiencing rapid growth in recent years, and numerous applications are improving the single-step efficiency of the whole assisted reproductive technology (ART) procedure. In this review, we collected all the algorithms supplying ART and selected those supporting the clinical assistance to the procedure up to the successful attempt. Those with a clear role in improving ART performances were further selected. We found a questionnaire-based algorithm identifying patients at risk for endometriosis with early management and better fertility outcome. An algorithm can detect the values of simple gamete production (male) and reservoir (female) according to gradual scale allocation, and display themas normal or abnormal, spontaneousor stimulated gamete production. This can provide significant benefits for infertile couples undergoing diagnostic and therapeutic journeys. The calculators for the starting dose of gonadotropins and the trigger timing during controlled ovarian stimulation make clinical management more efficient. With the application of AI in ART, the ability to determine the optimal number of metaphase II oocytes required for blastocyst formation and number of oocytes needed for embryo production has been significantly improved. The calculation of the implantation rate as proposed in different calculators, using the ultrasound of endometrial vascularization or the age and euploidy of the embryo transferred, may provide further advancement in managing the ART procedure with more participation from the couples to increase the efficacy of the procedures. Finally, the calculator of presumptive success with an ART program based on couples or medical center profiling and efficiency is of tremendous comfort to couples. In conclusion, algorithms and machine learning development in human reproduction are growing daily with evident benefits. Infertility treatments by in vitro fertilization (IVF) are assisted by several algorithms that improve the efficiency of each procedure step, making IVF program’s management more effortless.

Modeling Human Gonad Development in Organoids.

Our learning about human reproductive development is greatly hampered due to the absence of an adequate model. Animal studies cannot truthfully recapitulate human developmental processes, and studies of human fetal tissues are limited by their availability and ethical restrictions. Innovative three-dimensional (3D) organoid technology utilizing human pluripotent stem cells (hPSCs) offered a new approach to study tissue and organ development in vitro. However, a system for modeling human gonad development has not been established, thus, limiting our ability to study causes of infertility. In our study we utilized the 3D hPSC organoid culture in mini-spin bioreactors. Relying on intrinsic self-organizing and differentiation capabilities of stem cells, we explored whether organoids could mimic the development of human embryonic and fetal gonad. We have developed a simple, bioreactor-based organoid system for modeling early human gonad development. Male hPSC-derived organoids follow the embryonic gonad developmental trajectory and differentiate into multipotent progenitors, which further specialize into testicular supporting and interstitial cells. We demonstrated functional activity of the generated cell types by analyzing the expression of cell type-specific markers. Furthermore, the specification of gonadal progenitors in organoid culture was accompanied by the characteristic architectural tissue organization. This organoid system opens the opportunity for detailed studies of human gonad and germ cell development that can advance our understanding of sex development disorders. Implementation of human gonad organoid technology could be extended to modeling causes of infertility and regenerative medicine applications.

Transcriptional control of human gametogenesis.

ABSTRACTThe pathways of gametogenesis encompass elaborate cellular specialization accompanied by precise partitioning of the genome content in order to produce fully matured spermatozoa and oocytes. Transcription factors are an important class of molecules that function in gametogenesis to regulate intrinsic gene expression programs, play essential roles in specifying (or determining) germ cell fate and assist in guiding full maturation of germ cells and maintenance of their populations. Moreover, in order to reinforce or redirect cell fate in vitro, it is transcription factors that are most frequently induced, over-expressed or activated. Many reviews have focused on the molecular development and genetics of gametogenesis, in vivo and in vitro, in model organisms and in humans, including several recent comprehensive reviews: here, we focus specifically on the role of transcription factors. Recent advances in stem cell biology and multi-omic studies have enabled deeper investigation into the unique transcriptional mechanisms of human reproductive development. Moreover, as methods continually improve, in vitro differentiation of germ cells can provide the platform for robust gain- and loss-of-function genetic analyses. These analyses are delineating unique and shared human germ cell transcriptional network components that, together with somatic lineage specifiers and pluripotency transcription factors, function in transitions from pluripotent stem cells to gametes. This grand theme review offers additional insight into human infertility and reproductive disorders that are linked predominantly to defects in the transcription factor networks and thus may potentially contribute to the development of novel treatments for infertility.

Maternal phthalate exposure during pregnancy and male reproductive disorders: a systematic review and metaanalysis.

Phthalates are ubiquitous in the environment and they can penetrate the human body via multiple routes. However, the impact of phthalates on human male reproductive disorders remains unclear. A critical review of published studies was conducted to clarify the association of phthalates and male reproductive disorders and to highlight future research needs. PubMed, Cochrane Library, and Web of Science Database were systematically searched for relevant articles written in English, independent of region and time period. If more than one paper overlapped in study design or participants included, the most recent manuscript was included in our review. Due to limited homogeneous statistical data, observed trends were summarized to draw approximate conclusions. Nineteen manuscripts were included in our final analysis. Exposure to di-(2-ethylhexyl) phthalate (DEHP), di-n-butyl phthalate (DBP), diethyl phthalate (DEP), and/or benzyl butyl phthalate (BBP) is associated with a shorter anogenital distance (AGD). Meanwhile, exposure to DEHP and/or di-isodecyl phthalate (DIDP) is associated with higher risks for cryptorchidism and hypospadias. Generic exposure to phthalates has an adverse effect on human reproductive development, especially exposure to DEHP, DBP, DEP, BBP, and DIDP. A critical time for exposure sensitivity is during early pregnancy. Due to the lack of significant statistical power in this study, the conclusions drawn should be cautiously interpreted and they remain to be validated. Thus, additional well-designed studies, as well as propaganda and education regarding phthalate exposure and safer substitutes for these compounds, are greatly needed.

Investigation of the lncRNA THOR in Mice Highlights the Importance of Noncoding RNAs in Mammalian Male Reproduction.

THOR is a highly conserved testis-specific long noncoding RNA (lncRNA). The interaction between THOR and the development of the male reproductive system remains unclear. Herein, CRISPR/Cas9 technology was used to establish a stable THOR-deficient mouse model, and the relationship between THOR and the fertility of adult male mice was investigated. The male mice in which THOR was deleted were smaller than the WT male mice. Moreover, their survival rate was reduced by 60%, their fertility was reduced by 50%, their testicular size and sperm motility were reduced by 50%, their testicular cell apoptosis was increased by 7-fold, and their ratio of female-to-male offspring was imbalanced (approximately 1:3). Furthermore, to elucidate the mechanisms of male reproductive system development, the mRNA levels of THOR targets were measured by qRT-PCR. Compared with WT mice, the THOR-deficient mice exhibited significantly decreased mRNA levels of IGF2BP1, c-MYC, IGF1, and IGF2. MEK-ERK signaling pathway expression was downregulated as determined by Western blot. We found that THOR targeted the MER-ERK signaling pathway downstream of IGF2 by binding to IGF2BP1 and affected testicular and sperm development in male mice. These results may also provide perspectives for exploring the roles of lncRNAs in human reproductive development and the pathogenesis and potential therapeutic targets of infertility.

Studying human reproductive biology through single-cell analysis and in vitro differentiation of stem cells into germ cell-like cells.

Understanding the molecular and cellular mechanisms of human reproductive development has been limited by the scarcity of human samples and ethical constraints. Recently, in vitro differentiation of human pluripotent stem cells into germ cells and single-cell analyses have opened new avenues to directly study human germ cells and identify unique mechanisms in human reproductive development. The goal of this review is to collate novel findings and insightful discoveries with these new methodologies, aiming at introducing researchers and clinicians to the use of these tools to study human reproductive biology and develop treatments for infertility. PubMed was used to search articles and reviews with the following main keywords: in vitro differentiation, human stem cells, single-cell analysis, spermatogenesis, oogenesis, germ cells and other key terms related to these subjects. The search period included all publications from 2000 until now. Single-cell analyses of human gonads have identified many important gene markers at different developmental stages and in subpopulations of cells. To validate the functional roles of these gene markers, researchers have used the in vitro differentiation of human pluripotent cells into germ cells and confirmed that some genetic requirements are unique in human germ cells and are not conserved in mouse models. Moreover, transcriptional regulatory networks and the interaction of germ and somatic cells in gonads were elucidated in these studies. Single-cell analyses allow researchers to identify gene markers and potential regulatory networks using limited clinical samples. On the other hand, in vitro differentiation methods provide clinical researchers with tools to examine these newly identify gene markers and study the causative effects of mutations previously associated with infertility. Combining these two methodologies, researchers can identify gene markers and networks which are essential and unique in human reproductive development, thereby producing more accurate diagnostic tools for assessing reproductive disorders and developing treatments for infertility.

Developmental biology meets toxicology: contributing reproductive mechanisms to build adverse outcome pathways.

An adverse outcome pathway (AOP) is a simplified description of the sequence of mechanistic events that lead to a particular toxicological effect, from initial trigger to adverse outcome. Although designed to inform regulatory risk assessors, the AOP framework also provides a platform for innovative collaborations between experts from relevant research fields and the regulatory community. The underpinning for any AOP is basic knowledge about molecular and developmental processes; such knowledge can only be attained by solid bioscientific research. Starting with this fundamental knowledge, the objective is to devise novel testing strategies that focus on key events in a causative pathway. It is anticipated that such a knowledge-based approach will ultimately alleviate many of the burdens associated with classical chemical testing strategies that typically involve large-scale animal toxicity regimens. This hails from the notion that a solid understanding of the underlying mechanisms will allow the development and use of alternative test methods, including both in vitro and in silico approaches. This review is specifically targeted at professionals working in bioscientific fields, such as developmental and reproductive biology, and aims to (i) inform on the existence of the AOP framework and (ii) encourage new cross-disciplinary collaborations. It is hoped that fundamental biological knowledge can thus be better exploited for applied purposes: firstly, an improved understanding of how our perpetual exposure to environmental chemicals is causing human reproductive disease and, secondly, new approaches to screen for harmful chemicals more efficiently. This is not an instructional manual on how to create AOPs; rather, we discuss how to harness fundamental knowledge from the biosciences to assist regulatory toxicologists in their efforts to protect humans against chemicals that harm human reproductive development and function.

Atrazine induces penis abnormalities including hypospadias in mice.

Use of the herbicide atrazine (ATR) is banned in the European Union; yet, it is still widely used in the USA and Australia. ATR is known to alter testosterone and oestrogen production and thus reproductive characteristics in numerous species. In this proof of concept study, we examined the effect of ATR exposure, at a supra-environmental dose (5 mg/kg bw/day), beginning on E9.5 in utero, prior to sexual differentiation of the reproductive tissues, until 26 weeks of age, on the development of the mouse penis. Notably, this is the first study to specifically investigate whether ATR can affect penis characteristics. We show that ATR exposure, beginning in utero, causes a shortening (demasculinisation) of penis structures and increases the incidence of hypospadias in mice. These data indicate the need for further studies of ATR on human reproductive development and fertility, especially considering its continued and widespread use.

Effect of 2,3',4,4',5-Pentachlorobiphenyl Exposure on Endometrial Receptivity and the Methylation of HOXA10.

Polychlorinated biphenyls (PCBs) are one of the most common endocrine-disrupting chemicals and have obvious toxicity on human reproductive development. The aim of our study was to investigate the toxicity of chronic 2,3',4,4',5-pentachlorobiphenyl (PCB 118) exposure on embryo implantation and endometrial receptivity, with the possible mechanism of DNA methylation involved. Virgin CD-1 female mice (3 weeks old) were housed and orally treated with PCB 118 (0, 1, 10, 100 μg/kg) for a month. After mating with fertile males, the pregnant mice were killed on gestation day 4.5. Compared with the control group, implantation failures were observed in 1 μg/kg PCB 118- and 100 μg/kg PCB 118-treated groups. Abnormal endometrial morphology with open uterine lumens and densely compact stromal cells and poorly developed pinopodes were substantially in response to PCB 118 doses above, as well as the significant downregulation of implantation-associated genes (estrogen receptor 1, homeobox A10 [HOXA10], integrin subunit beta 3) and hypermethylation in the promoter region of HOXA10 further. It was confirmed that chronic exposure to PCB 118 produced an increased number of implantation failures in association with a defective uterine morphology during the implantation period. Alterations in methylation of HOXA10 could explain, at least in part, the mechanism of effects of PCB 118 exposure on the implantation process.

Melvin M. Grumbach 1925–2016

Generations of clinicians have been faced with often striking developmental and acquired hormonal syndromes associated with reproductive, growth, and metabolic phenotypes. These syndromes have included ambiguous genitalia, anomalies of sexual development, intersex forms, accelerated or delayed puberty, gigantism, and short stature. Although clinical phenotypes of these disorders have been well-established, elucidating their pathogenesis remained elusive until the mid-1950s. Drawing on his incisive application of novel diagnostic techniques to study neuroendocrine pituitary control, Mel Grumbach elucidated critical mechanisms underlying both normal physiology and pathogenesis of clinical syndromes, ranging from ontogeny of reproductive and growth axis control circuits, to effects of gonadotropin-releasing hormone (GnRH) pulse generation and timing of puberty onset, to control of adrenal androgen production and virilizing syndromes, to male estrogen receptor mutation sequelae. Melvin M. Grumbach. Image courtesy of Susan Merrell (University of California, San Francisco). Mel, an icon of investigative endocrinology, passed away on October 4, 2016, at the age of 90. Born, raised, educated, and trained as a physician, pediatrician, and endocrinologist in New York, Mel spent the latter 50 years of his stellar career at the University of California, San Francisco (UCSF), where he founded much of the knowledge base for our understanding of endocrine control of human reproductive development and growth. At the onset of his remarkable career of endocrine discovery in the mid-20th century, structural identification of peptide hormones and regulation of pituitary function were yet obscure. Hypothalamic hormonal control of normal and diseased human pituitary function was not yet characterized, assays for measuring hormones in the clinic were not sophisticated and were technically laborious, and mechanisms underlying hormone action mediated by receptor signaling were unknown (1). By the end of Mel’s career, he had both intimately witnessed the explosion of hormone discovery and also actively participated in the unprecedented knowledge spurt unraveling genetic … [↵][1]1Email: melmed{at}csmc.edu. [1]: #xref-corresp-1-1

Androgen Receptor Alternative Splicing and Human Infertility

About 95% of multi-exonic genes express more than one mRNA and downstream proteins by alternative splicing (AS) through the inclusion or exclusion of specific exons. Although AS provides a significant advantage in human evolution by increasing proteomic diversity, deregulation of AS can result in various pathologic conditions. The androgen receptor (AR), encoded by AR gene, is a steroid receptor transcription factor which mediates the cellular functions of androgen. The AR-mediated androgen actions play important and dual roles in the human reproduction development and function. Dysregulation of AR will result in human infertility. Multiple AR alternative splicing variants have been identified in different pathologies conditions, including androgen insensitivity syndrome, which will cause male infertility. More recently, our group has identified two AR splice variants expressed in granulosa cells from patients with polycystic ovary syndrome, which is one of the most common causes of female infertility. All of the aforementioned indicate that androgen receptor alternative splicing may be an important pathogenic mechanism in human infertility. The purpose of this review is to summarize the various alternatively spliced AR variants that have been discovered, with a focus on their role and origin in the pathologies of the human infertility diseases, including polycystic ovary syndrome and androgen insensitivity syndrome.

Measurement of sex steroids in murine blood and reproductive tissues by liquid chromatography–tandem mass spectrometry

Accurate measurement of sex steroids is essential to evaluate mouse models for human reproductive development and disorders. The recent advent of liquid chromatography–tandem mass spectrometry (LC–MS/MS) assays that match the sensitivity of steroid immunoassay could overcome problems arising from the limited specificity of steroid immunoassay. In this current study we validate a LC–MS/MS assay for the measurement of key sex steroids from murine serum and reproductive tissues. The assay gave excellent dilutional linearity (r2≥0.98) and reproducibility (CV≤10% of replicate samples) in serum and reproductive tissues with sensitive quantitation limits; testosterone (T; 2pg), dihydrotestosterone (DHT; 10pg), 5α-androstane-3α,17β-diol (3αDiol; 40pg), 5α-androstane-3β,17β-diol (3βDiol; 40pg), estradiol (E2; 0.5pg) and estrone (E1; 0.3pg). Using 0.1mL sample, T was the only consistently detectable steroid (detection limit 20pg/ml) in both male and female mouse serum. In the testis, T and DHT were quantifiable as were both diols at relatively high levels. Prostatic T levels were low and DHT was determined to be the most abundant androgen in this tissue. Uterine and ovarian levels of E2, E1 and T were measurable, with levels varying according to estrous cycle stage. Hence, we demonstrate that this LC–MS/MS method has the sensitivity, specificity and multi-analyte capability to offer accurate steroid profiling in mouse serum and reproductive tissues.

Effect of Light Perception on Menarche in Blind Women

ABSTRACTPurpose: The role of light on human reproductive development is unclear. Women with varying degrees of visual impairment appear to have altered reproductive function compared to sighted women. These differences have been attributed in part to differences in light exposure between the sighted and the blind. The present study was conducted to determine whether differences exist in reproductive measures among blind women with at least light perception (LP) compared to women with no perception of light (NPL). Methods: We studied 1,392 (LP, n = 968; NPL, n = 417; unreported, n = 7) blind women across North America between 2005 and 2007. Statistical analysis was conducted using Student's two-sample t tests and multivariate logistic or linear regression. Models were adjusted for current age, body mass index (BMI) and BMI at age 18. Results: NPL women reported an earlier menarche (mean age, 12.16, standard deviation ± 1.53) than LP women (mean age, 12.46, ± 1.57 yrs). The adjusted odds ratio (OR) for each increasing year of menarche among NPL women compared to LP women, was 0.88 (95% confidence intervals [CI]: 0.81–0.96). When those women NPL from birth were compared to all others, the adjusted odds ratio was strengthened (OR: 0.80, 95% CI: 0.68–0.94). When we examined the association between age at onset of NPL and age at menarche, we found a significant positive association with earlier menarche being associated with an earlier age category of loss of light perception (test for trend p < 0.01). Conclusions: Our findings suggest that lack of light perception affects reproductive development in women.

Mechanisms of action of phthalate esters, individually and in combination, to induce abnormal reproductive development in male laboratory rats

Phthalate esters are high production volume chemicals used to impart flexibility to polyvinyl chloride products as well as other applications. In the male laboratory rat, the period of sexual differentiation in utero is particularly sensitive to certain phthalate esters, which induce a suite of reproductive malformations, including epididymal and gubernacular agenesis. The fetal rat testes are a main target for phthalate esters as evidenced by a reduction in testosterone production and insulin-like hormone 3 (insl3) expression, a peptide hormone critical for testis descent. Histopathology of fetal and postnatal testes reveals that in utero exposure to phthalate esters disrupts Leydig and Sertoli cell maturation leading to a reduction in germ cells in the malformed seminiferous tubules in adulthood as well as an increased incidence of multinucleated germ cells. There are some strain-specific differences in the target organs in the male reproductive tract in rats affected by phthalate esters. Mixtures of phthalate esters with one another and with other anti-androgenic compounds exhibit cumulative, largely dose-additive effects on male reproductive tract development when administered during sexual differentiation in utero. Since phthalate ester metabolites are detected in maternal and fetal body fluids, and androgen-signaling and insl3 are highly conserved among mammals, phthalates may potentially affect human reproductive development.

Developmental plasticity of human reproductive development: Effects of early family environment in modern-day France

In a first study, we investigated how the absence of a father and the presence of a stepfather during early childhood affected physiological and behavioral traits related to reproductive development (such as age of menarche, age of first sexual intercourse and number of sexual partners) in a large sample set of male and female French university students. We evaluated which ages were sensitive to modifications in the family composition and found that menarche occurred earlier when the father was absent, particularly when the child was between 0 and 5 years of age. Father absence during early adolescence was associated with a younger age at first sexual intercourse and an increased number of sexual partners, for both sexes. The presence of a stepfather during this period further advanced the age of first sexual intercourse. We also measured testosterone levels in both sexes and analyzed their association with parental separation, and found that young women with separated parents had significantly higher afternoon levels of testosterone. In a second study, we analyzed direct fitness measures (such as number of children and grandchildren) in a large sample of French workers and found that parental separation during childhood was not associated with fitness variation. We discuss whether the reproductive outcomes of individuals having experienced modifications in the early family environment are the expression of costs or adaptive strategies.

The Problem of Species Comparison of Developmental Toxicity: Can We Extrapolate Human Developmental Toxicity Induced by Environmental Chemicals from the Data of Rodents?

Rodent models have great utility for evaluating the potential of environmental chemicals to alter human reproductive development. However, animal studies have some problems of species differences in extrapolating to human developmental toxicity induced by xenobiotics, because the placental endocrine functions in particular vary considerably among different species. For example, estrogen biosynthesis during pregnancy in humans is much different from that in rodents. In humans, ovarian function gradually declines after fertilization, as the placenta becomes the primary site of estrogen biosynthesis during pregnancy. In contrast to the process in humans, the ovary (not the placenta) is the main source of estrogen during pregnancy in rodents, because the placenta of rodents does not express the catalytic enzymes for estrogen biosynthesis, such as aromatase. The regulation of estrogen biosynthesis in the placenta is very important for human embryos because altering placental function can cause permanent effects on embryos. It has been suggested that rodents are therefore unsuitable for evaluating the potential effects of xenobiotics on the human reproductive system and developmental toxicity induced by the alteration of placental endocrine functions. Consequently, there is an urgent need to establish effective tools to evaluate the in vivo reproductive and developmental toxicity of environmental contaminants that disrupt the placental endocrine functions, including maintenance of local estrogen concentrations in the placenta. To resolve the problems, in this review we propose using transgenic mice, in which the transgene is controlled by placental-specific promoters, and local transgene systems into the placenta using viral vectors.

Guest Editorial: Assessing Risks and Benefits of Genistein and Soy

Guest Editorial: Assessing Risks and Benefits of Genistein and Soy

Relationship of Lead, Mercury, Mirex, Dichlorodiphenyldichloroethylene, Hexachlorobenzene, and Polychlorinated Biphenyls to Timing of Menarche Among Akwesasne Mohawk Girls

Relationship of Lead, Mercury, Mirex, Dichlorodiphenyldichloroethylene, Hexachlorobenzene, and Polychlorinated Biphenyls to Timing of Menarche Among Akwesasne Mohawk Girls