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Open access

Chinwe U Nwachukwu, Robert S Robinson, and Kathryn J Woad

Preovulatory follicle growth and the luteal transition require intense angiogenesis. This enables progesterone production to increase sufficiently to support a pregnancy. Inadequate follicular or luteal vascularisation can lead to reduced ovarian function and thus compromise fertility. Insulin-like growth factor 1 (IGF1) and IGF2 regulate multiple ovarian processes and are key links between an animal’s reproductive and metabolic status. This study investigated the role that the IGF system plays in regulating luteinising follicular endothelial cell (EC) networks and progesterone production in vitro. Bovine luteinising follicular angiogenesis cultures were treated with (i) LR3-IGF1 (10 or 100 ng/mL) under basal and angiogenic-stimulated conditions or (ii) IGF1 receptor (IGF1R) inhibitor (picropodophyllin (PPP); 1 µM) in the presence or absence of LR3-IGF1, IGF2 or combined LR3-IGF1 + IGF2 (10 ng/mL). EC networks were quantified by von Willebrand factor immunohistochemistry. Progesterone production was analysed by ELISA, and cell proliferation was determined by MTT assay. LR3-IGF1 had limited effects on EC growth parameters, whilst PPP (P < 0.001) markedly reduced EC growth parameters (by 60–70%). Cell proliferation was slightly increased (by 3–5%) by LR3-IGF1 (P < 0.001). LR3-IGF1 had variable effects on progesterone production, whilst PPP reduced progesterone concentration (P < 0.001) with or without LR3-IGF1 or IGF2 alone or in combination. IGF1 was detected in cell-conditioned media and was increased by LH (50 ng/mL) (P < 0.001). In conclusion, exogenous IGF1 and IGF2 had minimal effects on luteinising follicular angiogenesis and progesterone production, but the inhibitory effect of the IGF1R inhibitor (PPP) suggests that IGF1R signalling is critical for the development of EC networks and progesterone production in luteinising follicular cells.

Lay summary

The corpus luteum is a part of the ovary responsible for producing the critical pregnancy hormone, progesterone. To fulfil this function, the corpus luteum requires an extensive blood vessel network. Here, we investigated whether an important growth factor known to act on the ovary, insulin like growth factor (IGF) 1, critically regulates the formation of this blood vessel network and progesterone production. Cells from the corpus luteum were cultured with combinations of IGF1, a closely related hormone IGF2 and a chemical which stops both IGF1 and IGF2 from working. Afterwards, we measured the size and pattern of blood vessel networks, the production of progesterone and whether cells increased in number. We found adding IGF1 had limited effects, however stopping IGF1 from working had a very negative impact on both progesterone and on the formation of the blood vessel network. This suggests that cells from the corpus luteum were producing IGF1 and that IGF1/2 are critical for both blood vessel growth and hormone production.

Open access

Weizhou Wang, Mengmeng Zhao, Haiyang Zuo, Jingyao Zhang, Bin Liu, Fu Chen, Pengyun Ji, Guoshi Liu, Shuai Gao, Wei Shang, and Lu Zhang

Graphical Abstract

Abstract

The transition of maternal to zygotic gene expression regulation is critical for human preimplantation embryo development. In recent years, single-cell RNA sequencing (scRNA-seq) had been applied to detect the factors that regulate human oocyte maturation and early embryo development. Here, the evaluation of transcriptomes in single blastomere from the embryo collected from patients by scRNA-seq was performed. There were 20 blastomeres biopsied from 8-cell embryos of seven patients who received more than two ART cycles due to low embryo competence. Meanwhile, ten cells were collected from 8-cell embryos of four patients who received ART treatment due to male or tubal factors. The blastomeres were then evaluated using the previously established scRNA-seq method to determine the associations between their gene expression and developmental competence. The total number of genes detected in 8-cell embryos that failed to form blastocyst including maternal and zygotic mRNAs was reduced. There were 324 differently expressed genes detected among the 8-cell embryos including 65 genes that were significantly suppressed in the 8-cell embryos that failed to form blastocyst. Further analysis found these 8-cell embryos arrested at the cleavage stage due to the dysfunction of the cell cycle, DNA transcription activity, histone methylation, and cell division-related genes such as SMCO-1, ZNF271P,ZNF679, ASF1b, BEX3, DPPA2, and ORC4. The alterations of gene expression detected in human 8-cell embryos are tightly associated with its developmental competence and could be used as targets to enhance embryo development or parameters to predict the embryo’s development outcomes.

Lay summary

Many females are suffering infertility due to the failure of embryonic development at early stages due to unknown causes. At the very beginning of human embryo development, the embryos start to express its own genes, which should be achieved at 8-cell stage. In current research, we isolated one cell from 8-cell embryos and detected the gene expression at single-cell level. Then the remaining cells of these embryos were cultured to form blastocyst. Meanwhile, the data was analyzed according to the outcomes of embryo development. We detected 324 differently expressed genes between the 8-cell embryos that succeeded and failed to form blastocyst. Our research showed the association between the gene expression and the developmental competence of 8-cell embryos. The findings could be used to predict the embryo quality and potential therapy target to improve the efficiency of assisted reproductive techniques.

Open access

Lauren R Alesi, Quynh-Nhu Nguyen, Jessica M Stringer, Amy L Winship, and Karla J Hutt

Cytotoxic chemotherapies have been a mainstay of cancer treatment but are associated with numerous systemic adverse effects, including impacts on fertility and endocrine health. Irreversible ovarian damage and follicle depletion are the side effects of chemotherapy that can lead to infertility and premature menopause, both being major concerns of young cancer patients. Notably, many women will proceed with fertility preservation, but unfortunately existing strategies do not entirely solve the problem. Most significantly, oocyte and embryo freezing do not prevent cancer treatment-induced ovarian damage from occurring, which may result in the impairment of long-term hormone production. Unfortunately, loss of endogenous endocrine function is not fully restored by hormone replacement therapy. Additionally, while GnRH agonists are standard care for patients receiving alkylating chemotherapy to lessen the risk of premature menopause, their efficacy is incomplete. The lack of more broadly effective options stems, in part, from our poor understanding of how different treatments damage the ovary. Here, we summarise the impacts of two commonly utilised chemotherapies – cyclophosphamide and cis-diamminedichloroplatinum(II) (cisplatin) – on ovarian function and fertility and discuss the mechanisms underpinning this damage. Additionally, we critically analyse current research avenues in the development of novel fertility preservation strategies, with a focus on ferto-protective agents.

Lay summary

Over the past few decades, advances in the detection and treatment of cancer have dramatically improved survival rates in young women. This means that ensuring patients have a high quality of life after cancer treatment has become a new priority. Therefore, it is important to understand and prevent any long-term negative side effects of cancer treatments, with infertility and early-onset menopause being major concerns for women receiving chemotherapy. The current fertility preservation options available to young women have significant limitations. Therefore, the identification of new approaches to protect fertility has been an intense topic of research in recent years. In this review, we provide information on the negative side effects of two commonly used chemotherapy drugs – cyclophosphamide and cis-diamminedichloroplatinum(II) (cisplatin) – on fertility, and discuss how they cause damage to the ovaries. We also critically analyse recent preclinical studies related to the development of new fertility preservation techniques.

Open access

Agnes Stefansdottir, Magda Marečková, Magdalena Matkovic, Caroline M Allen, and Norah Spears

Females are born with a finite number of oocytes, collectively termed the ovarian reserve, established within the developing fetal ovary. Consequently, maternal exposure to reproductive toxicants can have harmful effects on the future fertility of her unborn female fetus. The chemical benzo[a]pyrene (B[a]P) is a prominent component of cigarette smoke. Despite it being a known ovotoxicant, around 8% of women in Europe smoke during pregnancy.

The purpose of this research was to examine the effect of B[a]P on the developing ovary, using the mouse as a model and with experiments carried out in vitro. B[a]P-exposure to the fetal ovary prior to follicle formation reduced the number of germ cells and subsequently, the number of healthy primordial follicles, by up to 76%; however, while proliferation of germ cells was not affected, the germ cells contained higher levels of DNA double-strand breaks. Exposure to B[a]P also affected the proportion of oocytes progressing through prophase I of meiosis. B[a]P exposure to neonatal mouse ovaries, after follicle formation, resulted in an 85% reduction in the number of healthy follicles, with a corresponding increase in apoptotic cell death and reduction in somatic cell proliferation. Although there was a trend towards a higher level of oxidative stress in B[a]P-exposed ovaries, this was not statistically significant; likewise, the antioxidant melatonin failed to protect against the B[a]P-induced ovarian damage. Together, the results here demonstrate that B[a]P-exposure damages the developing ovary, both before and shortly after follicle formation, an effect that could lead to a subsequent decrease in fertility.

Lay summary

Cigarette smoking during pregnancy can affect the fertility of the offspring, yet in Europe around 1 in 12 children born have been exposed to cigarette smoke before birth due to their mother smoking. Benzo[a]pyrene (B[a]P), one of the main chemicals found in cigarette smoke, can have damaging effects on the ovary as it develops in the fetus during the time that the population of future eggs, known as ovarian germ cells also develop. In this research, ovaries from mouse fetuses at stages of development, equivalent to the second and third trimesters of a human pregnancy, were cultured with or without B[a]P. Fetal mouse ovaries exposed to B[a]P had fewer germ cells and larger numbers of cells did not survive. Overall, the results suggest that development of the ovary of a fetus could be affected if the mother is exposed to B[a]P, whether that is through cigarette smoke, or other types of exposure.

Open access

Sarah Lensen, Sarah Armstrong, Emily Vaughan, Lucy Caughey, Michelle Peate, Cynthia Farquhar, Allan Pacey, Adam Balen, and Elaine Wainwright

In vitro fertilisation (IVF) add-ons are techniques, medicines, or procedures used in addition to standard IVF with the aim of improving the chance of success. The United Kingdom’s IVF regulator, the Human Fertilisation Embryology Authority (HFEA) developed a traffic light system to categorise add-ons as either green, amber, or red, based on results of randomised controlled trials. We undertook qualitative interviews to explore understanding and views of the HFEA traffic light system among IVF clinicians, embryologists, and IVF patients across Australia and the United Kingdom (n = 73). Overall, participants were supportive of the intention of the traffic light system; however, many limitations were raised. It was widely recognised that a simple traffic light system necessarily omits information which may be important to understanding the evidence. In particular, the red category was used in scenarios that patients viewed as having different implications for their decision-making, including ‘no evidence’ and ‘evidesssnce of harm’. Patients were surprised at the absence of any green add-ons and questioned the value of a traffic light system in this context. Many participants considered the website a helpful starting point, but desired more detail, including the contributing studies, results specific to patient demographics (e.g. <35 years and >35 years), and inclusion of more options (e.g. acupuncture). Overall, participants believed the website to be reliable and trustworthy, particularly due to the Government affiliation, and despite some concerns regarding transparency and an overly cautious regulator. The limitations of the traffic light system could be considered in any future updates to the HFEA website and others developing similar decision support tools.

Lay summary

In vitro fertilisation (IVF) add-ons are medical procedures or technologies that may be used in addition to standard IVF. They are usually used with the aim of increasing the chance of pregnancy and live birth. However, most add-ons have not been studied in high-quality clinical trials so it is uncertain whether they are beneficial. The UK’s IVF regulator developed a traffic light system for add-ons. They label them red, amber, or green, depending on whether there is evidence the add-on increases the chance of having a baby from IVF. We interviewed IVF patients, IVF doctors, and embryologists about the traffic light system. Overall, many people thought it was a reliable and trustworthy resource – however, many problems were identified. People generally thought the system was too simple and didn’t give enough information, it had limited detail about the number and types of studies included, and some important add-ons were missing, such as acupuncture.

Open access

Emily Bailie, Mila Maidarti, Robert Hawthorn, Stuart Jack, Neale Watson, Evelyn E Telfer, and Richard A Anderson

Androgens are essential in normal ovarian function and follicle health, but hyperandrogenism, as seen in polycystic ovary syndrome, is associated with disordered follicle development. There are few data on the effect of long-term exposure to high levels of testosterone as found in transgender men receiving gender-affirming endocrine therapy. In this study, we investigate the effect of testosterone on the development, morphological health and DNA damage and repair capacity of human ovarian follicles in vivo and their survival in vitro. Whole ovaries were obtained from transgender men (mean age: 27.6 ± 1.7 years; range: 20–34 years, n = 8) at oophorectomy taking pre-operative testosterone therapy. This was compared to cortical biopsies from age-matched healthy women obtained at caesarean section (mean age: 31.8 ± 1.5 years; range: 25–35 years, n = 8). Cortical tissues were dissected into fragments and either immediately fixed for histological analysis or cultured for 6 days and subsequently fixed. Follicle classification and morphological health were evaluated from histological sections stained with hematoxylin and eosin and expression of γH2AX as a marker of DNA damage by immunohistochemistry (IHC). In uncultured tissue, testosterone exposure was associated with reduced follicle growth activation, poor follicle health and increased DNA damage. After 6 days of culture, there was enhanced follicle activation compared to the control with further deterioration in morphological health and increased DNA damage. These data indicate that high circulating concentrations of testosterone have effects on the primordial and small-growing follicles of the ovary. These results may have implications for transgender men receiving gender-affirming therapy prior to considering pregnancy or fertility preservation measures.

Lay summary

As part of gender transitioning, transgender men take testosterone therapy. While androgens like testosterone are essential to maintain ovarian health, the effects of long-term testosterone treatment on the ovary are unclear. This study examines whether testosterone impacts ovarian follicle growth activation, follicle health and whether it causes DNA damage. It also looks at how well these follicles grow in tissue culture. The results showed there was a higher proportion of non-growing ovarian follicles in the ovaries of trans men, they appeared less healthy and there were higher levels of DNA damage. After 6 days of tissue culture, there were more growing follicles in transgender ovaries compared to control, but follicle health further deteriorated and there are increased levels of DNA damage. These results identify new effects of testosterone on the ovary and highlight the importance of discussing fertility preservation options prior to starting testosterone.

Open access

Sarah McCredie, Belinda An, Monika McShane, William Ledger, and Christos A Venetis

A prospective longitudinal cohort study aimed to longitudinally examine the kinetics of anti-Müllerian hormone (AMH) during the first two trimesters of pregnancy. Pregnant women with stored first-trimester serum samples were recruited at 24–28 weeks gestation during their gestational diabetes testing, where they provided an additional serum sample. The samples were analysed for AMH, oestradiol and progesterone concentrations. A decrease in serum AMH was observed in 40 out of 45 (88.9%) (95% CI: 75.9–96.3%) of the participants in this study. The median serum AMH concentration was 10.9 pmol/L in the first trimester and 6.5 pmol/L during the second trimester, with a significantly different distribution of the values between the first-trimester and the second-trimester AMH samples (P < 0.001). The median percentage of AMH difference of −39.8%. This study demonstrated a significant decrease in serum AMH levels from the first to the second trimester of pregnancy. The absolute decrease in AMH levels seems to be positively associated with first-trimester AMH levels, whereas the percentage of AMH difference is not. Further studies are required to elucidate the potential physiological mechanisms of this finding.

Lay summary

Anti-Müllerian hormone, also known as AMH, is produced by developing ovarian follicles in the ovary. The concentration of AMH in the serum is used as a marker of ovarian reserve. This marker has been shown to vary throughout the menstrual cycle and in women using hormonal contraception. This study examined this marker in women in the first and second trimesters of pregnancy to determine if it is variable throughout pregnancy. The study found that there was a significant decrease from the first to second trimester, with a larger decrease seen in women who had a higher first-trimester concentration of this marker. Further research is required to determine the physiological mechanism which causes the reduction of AMH in pregnancy.

Open access

Velez Carolina, Clauzure Mariangeles, Williamson Delia, Garcia Monica, Koncurat Mirta, and Barbeito Claudio

In the present work, we emphasize the studies about integrins and their receptors in pig placental interface at different times of gestation. Uterine placental interface (n = 24) of 17, 30, 60 and 70 days of gestation (dg) and non-pregnant uterus (n = 4) of crossbred sows were used. The presence of αvβ3 (ITGAV (integrin subunit alpha V) ITGB3 (integrin subunit beta 3)) and α5β1 (ITGA5 (integrin subunit alpha V) ITGB3 (integrin subunit beta 3)) integrins, and their ligands fibronectin (FN) and osteopontin (OPN/ SPP1), was detected by immunohistochemistry, and the immunolabeled area percentage (IAP) and the optical density (OD) were determined. The integrins and its ligands analyzed have presented peaks of expression in early and mid-gestation, both in IAP and in the OD area, decreasing at 70 dg. These temporal changes showed us that the molecules studied in this work participate in embryo/feto–maternal attachment, variably. Besides, we found a significant correlation both in the intensity and in the extension of immunostaining for trophoblastic FN and endometrial αvβ3, and trophoblastic OPN and endometrial α5β1, throughout the entire pig pregnancy. At late gestation, there is notable placental remodeling with subsequent removal or renewal of folds at the uterine–placental interface that results in the loss of focal adhesions. The decrease of the expression of some integrins and their ligands in late gestation, particularly at 70 dg, would demonstrate that there would be other adhesion molecules and other ligands that could be participating in the establishment of the maternal–fetal interface.

Lay summary

To carry a successful pregnancy, the formation of the placenta in pigs depends on adhesion molecules. Some of these molecules called integrins bind to other molecules such as fibronectin (FN) and osteopontin (OPN/SPP1). The variation in those molecule amounts during gestation would indicate which molecule is participating and what role it plays in pregnancy. We worked with pig placentas of early, mid- and late- gestation and non-pregnant uteruses. αvβ3 (ITGAV (integrin subunit alpha V) ITGB3 (integrin subunit beta 3)) and α5β1 (ITGA5 (integrin subunit alpha 5) ITGB1 (integrin subunit beta 1)) integrins, FN and OPN were found until mid-gestation but not at late gestation, suggesting that other types of molecules have a role in the last period of gestation.

Open access

Noof Abdulrahman Alrabiah, Constantine A Simintiras, Alexander C O Evans, Patrick Lonergan, and Trudee Fair

Follicular fluid (FF), a product of vascular transudate and granulosa and thecal cell secretions, is the milieu that has evolved to support oocyte growth and maturation which plays a central role in oocyte quality determination. Therefore, a suboptimal FF composition may be reflected in compromised oocyte progression through maturation, fertilization, or embryo development. To date, the composition of bovine FF remains understudied. To address this, we comprehensively characterized the metabolomic constituency of bovine FF in the period during which the oocyte undergoes meiotic maturation. More specifically, FF from pre (−24 h) and peri (−2 h)-ovulatory follicles was profiled by high-throughput untargeted ultra-HPLC tandem mass spectroscopy. A total of 634 metabolites were identified, comprising lipids (37.1%), amino acids (30.0%), xenobiotics (11.5%), nucleotides (6.8%), carbohydrates (4.4%), cofactors and vitamins (4.4%), peptides (3.6%), and energy substrates (2.1%). The concentrations of 67 metabolites were significantly affected by the stage of follicle development, 33.3% (n = 21) were reduced (P ≤ 0.05) by a mean of 9.0-fold, whereas 46 were elevated (P ≤ 0.05) by a mean of 1.7-fold in peri- vs pre-ovulatory FF. The most pronounced individual metabolite concentration decreases were observed in hypoxanthine (98.9-fold), xanthine (65.7-fold), 17β-oestradiol (12.4-fold), and inosine (4.6-fold). In contrast, the greatest increases were in retinal (4.9-fold), 1-methyl-5-imidazoleacetate (2.7-fold), and isovalerylcarnitine (2.7-fold). This global metabolomic analysis of bovine FF temporal dynamics provides new information for understanding the environment supporting oocyte maturation and facilitating ovulation that has the potential for improving oocyte quality both invivo and in vitro.

Lay Summary

The ovaries are part of the female reproductive system, and they produce and store eggs in structures known as ‘follicles’. Depending on the species, one or more follicles release an egg from the ovary during ovulation. FF, which is formed from the secretions of follicle cells and substances delivered from the bloodstream, bathes the eggs as they develop within their follicles. For pregnancy to happen, the egg must be capable of being fertilised by a sperm cell, developing into an embryo and implanting it in the womb. FF has evolved to support the egg to achieve this. Using the cow as a model, this study looks at the composition of FF during the final hours before ovulation, when the egg becomes mature and ready for fertilisation. More than 600 different substances were identified, providing new information, that has the potential to improve egg quality.