Controlled ovarian stimulation (COS) is a major component of assisted reproductive technologies. Clinically, it has been observed that some women experience changes in thyrotropin levels following COS, which then bring about subclinical hypothyroidism and may adversely affect conception. Studies have also shown that the specific degree as well as the tendency of changes in thyroid function vary with differences in thyroid function before pregnancy, thyroid autoimmunity, the COS regimen, and the observation time point. However, the associated pathophysiological mechanism of the effects of COS on pregnancy has not yet been fully elucidated. This may be because increased estradiol levels, caused by COS, induce increased levels of thyroxine-binding globulin, resulting in a decrease in free thyroxine (FT4) level and an increase in TSH level. Conversely, it has also been observed that human chorionic gonadotropin (hCG) can act directly on thyroid cells, exerting opposite effects on FT4 and TSH levels. Additionally, the effects of COS on thyroid function may be more pronounced, especially in women with autoimmune thyroid disease or thyroid dysfunction before pregnancy, ultimately leading to subclinical hypothyroidism. Here, we review recent research progress regarding the effects of COS on thyroid function during pregnancy.

Summary Sentence

The use of controlled ovarian stimulation drugs in assisted reproductive technologies can increase serum thyrotropin levels, possibly leading to hypothyroidism.

Introduction: Cisplatin-based chemotherapy is the standard cancer therapy; however, this treatment causes depletion of ovarian follicles in women of reproductive age. Adjuvant treatment with melatonin can protect the ovaries from oxidative stress, reducing the side effects of chemotherapy. The objective was to evaluate the effects of the use of melatonin on the ovarian follicles of mice treated with cisplatin. Methodology: A systematic review was performed. The search strategy used the terms: “cisplatin”, “melatonin,” and “ovarian”. MEDLINE EMBASE, Cochrane Library, and gray literature (Google Scholar) were used as databases. The search was limited to experimental studies, performed on animals, with no language restrictions. Results: The search identified 30 studies and 5 primary studies, published between 2016 and 2021, and met the inclusion criteria, with a total of 115 mice. For the p-FOX3a/FOXO3a pathway, the meta-analysis showed a standard mean difference (SMD) of –4.79 (95% CI –6.16 to –3.42; P < 0.00001, two studies, 38 mice; I² = 0%). For the p-PTEN pathway, the meta-analysis showed an SMD of –1.65 (95% CI –2.71 to –0.59; P = 0.002, two studies, 38 mice; I² = 47%). Conclusion: Melatonin variation in efficacy varies according to the dose used in mice previously exposed to cisplatin. However, melatonin was able to alter the p-PTEN and p-FOX3a/FOXO3a pathways.

Summary Sentence

Melatonin interfered with the p-PTEN and p-FOX3a/FOXO3a pathways and Cisplatin associated with melatonin showed a reduction in ovarian follicles loss.

Citrullination, the post-translational modification of arginine residues, is catalyzed by the four catalytically active peptidylarginine deiminase (PAD or PADI) isozymes and alters charge to affect target protein structure and function. PADs were initially characterized in rodent uteri and, since then, have been described in other female tissues including ovaries, breast, and the lactotrope and gonadotrope cells of the anterior pituitary gland. In these tissues and cells, estrogen robustly stimulates PAD expression resulting in changes in levels over the course of the female reproductive cycle. The best-characterized targets for PADs are arginine residues in histone tails, which, when citrullinated, alter chromatin structure and gene expression. Methodological advances have allowed for the identification of tissue-specific citrullinomes, which reveal that PADs citrullinate a wide range of enzymes and structural proteins to alter cell function. In contrast to their important physiological roles, PADs and citrullinated proteins are also involved in several female-specific diseases including autoimmune disorders and reproductive cancers. Herein, we review current knowledge regarding PAD expression and function and highlight the role of protein citrullination in both normal female reproductive tissues and associated diseases.

Summary Sentence

Peptidylarginine deiminase enzymes are expressed in female reproductive tissues and post-translationally convert arginine residues in target proteins to citrulline.

Graphical Abstract

The pig is an excellent animal model for simulating human physiology and a major animal for meat production and xenotransplantation. Therefore, researching porcine embryonic development is crucial for studying human reproductive diseases and improving litter size in commercial pigs. Embryonic development in pigs occurs under a complex regulatory mechanism, in which epigenetic regulatory mechanisms play an essential role. Recently, studies on the effects of epigenetic modifications on embryonic development have been conducted at different developmental stages and in different cell lines. Increasing evidence suggests that a certain amount of crosstalk exists between different epigenetic modifications. This review describes four regulatory mechanisms of epigenetics involved in porcine embryonic development: DNA methylation, histone modification, non-coding RNA function, and chromatin accessibility, and explores the possible crosstalk between them.

Summary Sentence

In this review, four epigenetic modifications (DNA methylation, histone modification, non-coding RNA function, and chromatin accessibility) were described in porcine embryonic development, and the possible crosstalk between them was explored in order to provide references for further understanding and studying the role of epigenetic modification in embryonic development.

Linker histone H1 binds to the nucleosome and is implicated in the regulation of the chromatin structure and function. The H1 variant H1FOO is heavily expressed in oocytes and early embryos. However, given the poor homology of H1FOO among mammals, the functional role of H1FOO during preimplantation embryonic development remains largely unknown, especially in domestic animals. Here, we find that H1FOO is not only expressed in oocytes and preimplantation embryos but granulosa cells and spermatids in cattle. We then demonstrate that the interference of H1FOO results in preimplantation embryonic developmental arrest in cattle using either RNA editing or Trim-Away approach. H1FOO depletion leads to a compromised expression of critical lineage-specific genes at the morula stage and affects the establishment of cell polarity. Interestingly, H1FOO depletion causes a significant increase in the expression of genes encoding other linker H1 and core histones. Concurrently, there is an increase of H3K9me3 and H3K27me3, two markers of repressive chromatin and a decrease of H4K16ac, a marker of open chromatin. Importantly, overexpression of bovine H1FOO results in severe embryonic developmental defects. In sum, we propose that H1FOO controls the proper chromatin structure that is crucial for the fidelity of cell polarization and lineage specification during bovine preimplantation development.

Egg activation in mammals is triggered by oscillations in egg intracellular calcium (Ca²⁺) level. Ca²⁺ oscillation patterns can be modified in vitro by changing the ionic composition of culture media or in vivo by conditions affecting mitochondrial function, such as obesity and inflammation. In mice, disruption of Ca²⁺ oscillations in vitro impacts embryo development and offspring growth. Here we tested the hypothesis that, even without in vitro manipulation, abnormal Ca²⁺ signaling following fertilization impacts offspring growth. Plasma membrane Ca²⁺ ATPases (PMCA) extrude cytosolic Ca²⁺ to restore Ca²⁺ homeostasis. To disrupt Ca²⁺ signaling in vivo, we conditionally deleted PMCA1 (cKO) in oocytes. As anticipated, in vitro fertilized cKO eggs had increased Ca²⁺ exposure relative to controls. To assess the impact on offspring growth, cKO females were mated to wild type males to generate pups that had high Ca²⁺ exposure at fertilization. Because these offspring would be heterozygous, we also tested the impact of global PMCA1 heterozygosity on offspring growth. Control heterozygous pups that had normal Ca²⁺ at fertilization were generated by mating wild type females to heterozygous males; these control offspring weighed significantly less than their wild type siblings. However, heterozygous offspring from cKO eggs (and high Ca²⁺ exposure) were larger than heterozygous controls at 12 week-of-age and males had altered body composition. Our results show that global PMCA1 haploinsufficiency impacts growth and support that abnormal Ca²⁺ signaling after fertilization in vivo has a long-term impact on offspring weight. These findings are relevant for environmental and medical conditions affecting Ca²⁺ handling and for design of culture conditions and procedures for domestic animal and human assisted reproduction.

Summary Sentence

Plasma membrane calcium ATPase 1 (PMCA1) in eggs regulates calcium homeostasis at fertilization, and offspring derived from PMCA1-null eggs (and excess calcium signal at fertilization) weigh more than controls with normal calcium.

Graphical Abstract

After fertilization, the zygote undergoes cell division. Up to the 8-cell stage, the blastomeres of mouse preimplantation embryos are morphologically identical. The first cell differentiation starts in the morula leading to the formation of trophectoderm cells and inner cell mass cells of the blastocyst. The regulation of the differentiation event and the formation of blastocysts are not fully known. Lethal-7 (let-7) is a family of evolutionarily conserved microRNAs. Here, we showed that the expression of let-7a and let-7g decreased drastically from the 1-cell stage to the 2-cell stage, remained low up to the 8-cell stage and slightly increased after the morula stage of mouse embryos. The expression of let-7 in the inner cell mass was higher than that in the trophectoderm. Forced expression of let-7a in embryos at the 1-cell and 4-cell stage inhibited blastocyst formation and downregulated the expression of CDX2 but maintained that of OCT4 in the trophectoderm. Forced expression of other let-7 isoforms exhibited similar inhibitory action on blastulation. On the other hand, inhibition of let-7a at the 4-cell stage and the 8-cell stage enhanced blastocyst formation. Co-injection of green fluorescent protein (GFP) mRNA (lineage tracer) with either precursor of let-7a (pre-let-7a) or scramble control into one blastomere of 2-cell embryos showed that ∼75% of the resulting blastocysts possessed GFP⁺ cells in their inner cell mass only. The biased development towards the inner cell mass with forced expression of let-7 was reproduced in 2-cell chimeric embryos consisting of one wildtype blastomere and one GFP mRNA-injected blastomere from another 2-cell embryo carrying a doxycycline-inducible let-7g gene. Bioinformatics analysis indicated that Tead4 was a potential target of let-7. Let-7 bound to the 3′UTR of Tead4 and let-7 forced expression downregulated the expression of Tead4 in mouse blastocysts. Co-injection of Tead4 mRNA partially nullified the modulatory roles of let-7a in the inner cell mass cell fate. In conclusion, a high level of let-7 at the 2-cell stage favored the formation of the inner cell mass, whereas a low level of let-7 at the 4-cell to 8-cell stage enhanced blastocyst formation. Tead4 mediated the action of let-7 on the inner cell mass cell-fate determination.

Graphical Abstract

Uterine endometrial differentiation is essential for developmental continuity and female health. A convenient in vitro model mimicking the physiological status is needed to effectively evaluate implantation and uterine response mechanisms. Thus, we developed a promising in vitro model, the FSS (FSH mimic-stimulated synchronized) model, by using primary mouse uterine stromal cells (mUSCs) obtained from equine chorionic gonadotropin (eCG)-primed mice. These mUSCs could be differentiated into decidualized cells with 17 beta-estradiol (E2) and progesterone (P4). The pregnancy day 4 (PD4) model, in which mUSCs are obtained at day 4 of pregnancy, was used as a control. The cell shape index and polyploidy rates were similar between the two models. The staining intensities of lipids and glycogen were significantly higher in the induced groups in both models but stronger in the FSS model than in the PD4 model. The expression levels of AP-TNAP, cathepsin L, Prl8a2, Gja1, Cebpb, and Igfbp1 were increased at 24 h after decidual induction. PR-alpha and PR-beta levels were also increased at 24 h after decidual induction in both models. These results indicate that the FSS model provides a convenient method for obtaining USCs that are usable for various experimental approaches due to their physiological competence and flexibility for triggering induction. This may serve as a model system for the study of pathogeneses originating from the endometrium or communication with other tissues and lead to a better understanding of embryo implantation mechanisms. Furthermore, the results of this study will be integral for further refinements of 3D uterine culture manipulation techniques.

Summary Sentence

An artificial in vitro decidual induction model with physiologically competent stromal cells is presented to facilitate experimental designs. This will help deepen the study of embryo implantation and aid in exploration of the causes of implantation-related diseases.

Graphical Abstract

Morphogenesis of the female reproductive tract is regulated by the mesenchyme. However, the identity of the mesenchymal lineage that directs the morphogenesis of the female reproductive tract has not been determined. Using in vivo genetic cell ablation, we identified Amhr2+ mesenchyme as an essential mesenchymal population in patterning the female reproductive tract. After partial ablation of Amhr2+ mesenchymal cells, the oviduct failed to develop its characteristic coiling due to decreased epithelial proliferation and tubule elongation during development. The uterus displayed a reduction in size and showed decreased cellular proliferation in both epithelial and mesenchymal compartments. More importantly, in the uterus, partial ablation of Amhr2+ mesenchyme caused abnormal lumen shape and altered the direction of its long axis from the dorsal-ventral axis to the left–right axis (i.e., perpendicular to the dorsal-ventral axis). Despite these morphological defects, epithelia underwent normal differentiation into secretory and ciliated cells in the oviduct and glandular epithelial cells in the uterus. These results demonstrated that Amhr2+ mesenchyme can direct female reproductive tract morphogenesis by regulating epithelial proliferation and lumen shape without affecting the differentiation of epithelial cell types.

Summary Sentence

Amhr2+ mesenchyme regulates morphogenesis of the female reproductive tract along antero-posterior and dorso-ventral axes.

The dromedary camel (Camelus dromedarius) is a short-day desert breeder in which female ovulation is induced by mating. Current data indicate that male-induced ovulation is triggered by its seminal plasma nerve growth factor beta (β-NGF), but the exact mechanisms involved in the induction of ovulation are still unknown. In this study, we report that an intramuscular injection of β-NGF in sexually active short-day-adapted female camels induces an ovulation attested by a surge of circulating LH (2–6 h after treatment) followed by an oocyte release with its cumulus oophorus (confirmed by ultrasonography 72 h after treatment) and a large and progressive increase in circulating progesterone (significant from the 2nd to the 10th days after β-NGF injection). In addition, this β-NGF treatment induces a broad nuclear c-FOS activation in cells located in various hypothalamic areas, notably the preoptic area, the arcuate nucleus, the dorso- and ventromedial hypothalamus, the paraventricular nucleus, and the supraoptic nucleus. A double immunostaining with neuropeptides known to be involved in the central control of reproduction indicates that ∼28% kisspeptin neurons and 43% GnRH neurons in the proptic area, and ∼10% RFRP-3 neurons in the dorso- and ventromedial hypothalamus are activated following β-NGF injection. In conclusion, our study demonstrates that systemic β-NGF induces ovulation in the female dromedary camel and indicates that this effect involves the central activation of hypothalamic neurons, notably the kisspeptin neurons.

Summary Sentence

In conclusion, our study demonstrates that systemic β-NGF induces ovulation in the female dromedary camel and indicates that this effect involves the central activation of hypothalamic neurons, notably the kisspeptin neurons.

Graphical Abstract

A molecular interaction between maternal endometrium and implanting conceptus can lead to activation of a variety of transcription factors that regulate expression of several genes necessary for the process of embryo implantation. While, signal transducer and activator of transcription 3 (STAT3) is responsible for decidualization and epithelial remodeling in humans and mice, its role in porcine endometrium has not been explored before. In the present study, we observed a pregnancy dependent increase in gene and protein expression of STAT3. Phosphorylated STAT3 was predominantly present in the endometrium of pregnant animals in luminal and glandular epithelium and in the endothelium of blood vessels with a weak staining in stromal cells. Interleukins, IL-1β and IL-6, and epidermal growth factor (EGF)-induced STAT3 expression and phosphorylation in endometrial explants collected on Day 13 of the estrous cycle. Biological significance of STAT3 was evaluated by blocking its phosphorylation with STAT3-specific inhibitor, Stattic. Using porcine extracellular matrix (ECM) and adhesion molecule array, EGF was shown to induce changes in gene expression of ECM components: MMP1, MMP3, MMP12, LAMA1, SELL, and ICAM1, which was abrogated in the presence of Stattic. Transcriptional activity of STAT3 was observed in promoter regions of MMP3 and MMP12. Additionally, IL-6-induced STAT3 phosphorylation upregulated VEGF and VCAM1 abundances in endometrial-endothelial cells (EEC). Moreover, IL-6 resulted in an increase in EEC proliferation and capillary formation which was reversed in the presence of Stattic. Results of present study reveal a role for STAT3 phosphorylation in regulating extracellular matrix remodeling and angiogenesis in porcine endometrium to facilitate embryo implantation.

Summary Sentence

Cytokines IL-1β, IL-6, and the growth factor, epidermal growth factor activate signal transducer and activator of transcription 3, resulting in its nuclear translocation in the porcine endometrial cells to induce changes in extracellular matrix components and endometrial angiogenesis during implantation period.

Graphical Abstract

Identification of placental dysfunction in early pregnancy with noninvasive imaging could be a valuable tool for assessing maternal and fetal risk. Dynamic contrast enhanced (DCE) magnetic resonance imaging (MRI) can be a powerful tool for interrogating placenta health. After inoculation with Zika virus or sham inoculation at gestation age (GA) 45 or 55 days, animals were imaged up to three times at GA65, GA100, and GA145. DCE MRI images were acquired at all imaging sessions using ferumoxytol, an iron nanoparticle-based contrast agent, and analyzed for placental intervillous blood flow, number of perfusion domains, and perfusion domain volume. Cesarean section was performed at GA155, and the placenta was photographed and dissected for histopathology. Photographs were used to align cotyledons with estimated perfusion domains from MRI, allowing comparison of estimated cotyledon volume to pathology. Monkeys were separated into high and low pathology groups based on the average number of pathologies present in the placenta. Perfusion domain flow, volume, and number increased through gestation, and total blood flow increased with gestation for both low pathology and high pathology groups. A statistically significant decrease in perfusion domain volume associated with pathology was detected at all gestational ages. Individual perfusion domain flow comparisons demonstrated a statistically significant decrease with pathology at GA100 and GA145, but not GA65. Since ferumoxytol is currently used to treat anemia during human pregnancy and as an off-label MRI contrast agent, future transition of this work to human pregnancy may be possible.

Summary Sentence

Ferumoxytol dynamic contrast-enhanced MRI can detect changes in placental perfusion associated with term placental pathology after Zika infection in early gestation.

Graphical Abstract

Ruminant conceptuses that elongate and attach to the uterine luminal epithelium (LE) to establish pregnancy require a large amount of adenosine triphosphate (ATP). The creatine (Cr)–creatine kinase (CK)–phosphocreatine (PCr) system re-generates ATP in dividing and migrating cells such as the conceptus trophectoderm cells. However, little is known about metabolism of Cr within uterine and conceptus tissues in livestock species during early gestation. In this study, Suffolk ewes were ovariohysterectomized on Days 9, 12, 15, 16, 17, 18, 20, or 21 of pregnancy (n = 2–5 animals/per day) to investigate metabolites, mRNAs, and proteins of the Cr–CK–PCr system at uterine–conceptus interface. Amounts of Cr and guanidinoacetate (GA) in uterine flushings increased between Days 12 and 17 of pregnancy. Endometrial expression of mRNAs for GA formation (AGAT), Cr synthesis (GAMT), and Cr/PCr utilization (CKB) was greater on Days 17 and 21 than on Days 9 and 12 of pregnancy. Immunoreactive AGAT was detected in uteri only on Day 21 but not in uteri or conceptuses at earlier days of pregnancy. GAMT, SLC6A8, and CKs were expressed in uterine luminal and glandular epithelia. Immunoreactive CKs (CKB, CKM, and CKMT1) appeared greater on Day 9 than Day 17 of pregnancy. Immunoreactive GAMT and CKs appeared greater in trophectoderm of conceptuses on Day 20 than on Day 15 of pregnancy, whereas the opposite was observed for that of SLC6A8. This study provides insights into cell-, tissue-, and time-specific metabolism of Cr at the uterine–conceptus interface suggesting a role for the Cr–CK–PCr system in ovine conceptus development and implantation.

Summary Sentence

Creatine is abundant in the histotroph, and the proteins required for synthesis and transport of creatine are expressed in uteri and conceptuses during early gestation in sheep indicating a role for creatine metabolism in conceptus development.

Uterine contraction is crucial for a successful labor and the prevention of postpartum hemorrhage. It is enhanced by hypoxia; however, its underlying mechanisms are yet to be elucidated. In this study, transcriptomes revealed that hypoxia-inducible factor-1alpha was upregulated in laboring myometrial biopsies, while blockade of hypoxia-inducible factor-1alpha decreased the contractility of the myometrium and myocytes in vitro via small interfering RNA and the inhibitor, 2-methoxyestradiol. Chromatin immunoprecipitation sequencing revealed that hypoxia-inducible factor-1alpha directly binds to the genome of contraction-associated proteins: the promoter of Gja1 and Ptgs2, and the intron of Oxtr. Silencing the hypoxia-inducible factor-1alpha reduced the expression of Ptgs2, Gja1, and Oxtr. Furthermore, blockade of Gja1 or Ptgs2 led to a significant decrease in myometrial contractions in the hypoxic tissue model, whereas atosiban did not remarkably influence contractility. Our study demonstrates that hypoxia-inducible factor-1alpha is essential for promoting myometrial contractility under hypoxia by directly targeting Gja1 and Ptgs2, but not Oxtr. These findings help us to better understand the regulation of myometrial contractions under hypoxia and provide a promising strategy for labor management and postpartum hemorrhage treatment.

Methods for standard in vitro fertilization have been difficult to establish in the horse. We evaluated whether prolonged sperm pre-incubation would support subsequent fertilization. Fresh sperm were pre-incubated with penicillamine, hypotaurine, and epinephrine (PHE) for 22 h. Co-incubation of cumulus-oocyte complexes (COCs) for 6 h yielded 43% fertilization; culture of presumptive embryos yielded 21% blastocysts. Sperm incubated similarly, but without PHE, did not fertilize oocytes. Use of extended semen in the system yielded 54% blastocysts and was applied in subsequent experiments. Transfer of three in vitro fertilization-produced blastocysts to recipient mares resulted in birth of three normal foals. When sperm were pre-incubated for 22 h, 47–79% of oocytes were fertilized after 1 h of co-incubation. Sperm pre-incubated for 15 min or 6 h before co-incubation yielded no fertilization at 1 h, suggesting that capacitation in this system requires between 6 and 22 h. Sperm assessed after 15 min, 6 h, or 22 h pre-incubation showed increasing protein tyrosine phosphorylation of the midpiece, equatorial band, and apical head; this pattern differed from that induced by high pH conditions and may denote functional equine sperm capacitation. Use of the final devised system, i.e., extended semen, with 22 h of sperm pre-incubation and 3 h of COC co-incubation, yielded 90% fertilization with a blastocyst rate of 74%. This is the first report of efficient and repeatable standard in vitro fertilization in the horse and the first report of in vitro production of blastocysts and resulting foals after in vitro fertilization.

Summary Sentence Pre-incubation of fresh equine sperm for 22 h in the presence of penicillamine, hypotaurine and epinephrine supports high rates of in vitro fertilization and production of viable blastocysts.

Sertoli cells (SCs), the only somatic cells in the seminiferous tubules, facilitate the maintenance of testicular immune privilege through the formation of the blood-testis barrier (BTB) and the expression of immunoregulatory factors. Rho guanosine exchange factor 15 (ARHGEF15) is a member of the guanosine exchange factors, which are involved in cell migration, cell polarity, and cell cycle progression via activation of Rho GTPases. This study investigated the functional role of ARHGEF15 in SCs during spermatogenesis using SC–specific Arhgef15 knockout mice. The results revealed that Arhgef15 deficiency in SCs affected the localization of SC nuclei, disrupted BTB integrity, and led to premature shedding of germ cells. In Arhgef15^flox/flox/Amh-Cre⁺ mice, the ultrastructure of the round spermatids was impaired, accompanied by acrosome degeneration, acrosomal vesicle shedding, and atrophic nuclei. Consequently, the percentage of abnormal sperm in the Arhgef15^flox/flox/AmhCre⁺ epididymis was markedly elevated. RNA-sequencing analysis revealed that most of the differentially expressed genes in SCs of Arhgef15^flox/flox/Amh-Cre⁺ mice were associated with immunity. Further study revealed that the sera of Arhgef15^flox/flox/Amh-Cre⁺ mice showed immunoreactivity against testicular lysate of wild-type mice, indicating the production of antibodies against testicular autoantigens in Arhgef15^flox/flox/Amh-Cre⁺ mice. In conclusion, the specific deletion of Arhgef15 in SCs of mice leads to sperm abnormality, probably by disrupting the testicular immune homeostasis.

Summary Sentence

Guanylate exchange factor Arhgef15 is required for the development of germ cells by maintaining the balance of immune-related factors in mouse testes.