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Danièle Klett and Yves Combarnous
In previous studies, we had shown the synergistic effect of 10−5 M forskolin (FSK) on the detection threshold of the cyclic AMP response to luteinizing hormones (LH) and chorionic gonadotropins (CG) from various species in the mouse Leydig tumor cell (mLTC) cell line. Independently, we started to study the effect of 10−12–10−6 M oxytocin (OXT) also on the cyclic AMP response to LH and CG preparations on these same cells and found an amplifying effect on the luminescence response caused by gonadotropins. The aim was then to explore the effects of 10−12–10−6 M OXT on the gonadotropin-induced cAMP response, in the presence or absence of 10 µM FSK to optimize the assay down to a sensitivity compatible with the detection of the circulating concentrations of these hormones in various species. Finally, the optimization relies on three independent phenomena: (1) the inhibition of nucleotide phosphodiesterase by IBMX (3-isobutyl-1-methylxanthine) to avoid cAMP degradation; (2) the strong synergy of 10 µM forskolin with low concentrations of LH or CG during the 1-h luminescence measurement; (3) the stimulatory effect of 10−8M OXT on the amplitude of transfected cAMP-sensitive luciferase response. By doing this, the detectable concentrations are at the 1–10 pg/well (pM range) for the LHs and CGs from various species. The bioactivities of circulating LHs and CGs in blood or urine are therefore expected to be measurable in 10 µL-plasma samples from mammalian species and maybe others. Indeed, a preliminary study with equine and donkey plasma samples shows that the measured bioactivity was fully inhibited by a specific MAB against the receptor-binding region of equine LH (eLH) and equine CG (eCG), thus eliminating a possible response due to interfering substances other than eLH or eCG. From these data, it is expected that the bioactivity profiles of these hormones will be measurable in the blood of human, equine, and ovine species and very likely in rodents, ruminants, and hopefully in most other mammalian species.
Lay summary
Luteinizing hormone (LH) plays a central role in controlling ovary and testicle functions in many animals, including humans. The highly sensitive method, known as an assay, described in this paper, measures the biological activity of LH in the blood of mammals. The assay is performed in culture of cells derived from mouse testicles in the presence of factors that diminish the detection threshold for LH. The knowledge of the bioactive LH concentration dynamics in the blood is very informative about the reproductive status of male and female mammals. This new in vitro bioassay provides a powerful tool to get this information.
Clara Malo, Sara Carracedo, Maryse Delehedde, Nicolas Sergeant, and Julian Alexandra Skidmore
ProAKAP4 is synthetized as a precursor polypeptide that must be converted into mature AKAP4 in living spermatozoa and is considered as a functional marker of spermatozoa. The gene is well-conserved in mammals although uncharacterized in Camelidae. In the present study, we investigate the expression metabolism of proAKAP4 and AKAP4 proteins and evaluate their seasonal dynamics relative to semen quality in dromedary camels. Semen parameters including volume and viscosity and characteristics of sperm including concentration, total production, total and progressive motility, vitality, acrosome integrity and morphological abnormalities were assessed in semen samples collected weekly from six camels during the rutting season, from November to April. Only total sperm production varied, peaking in January. Both the precursor proAKAP4 and AKAP4 proteins were investigated and shown to express biochemical properties similar to those described in other mammals. ProAKAP4 concentrations expressed in ng/10 million spermatozoa as assayed using a specific ELISA showed a strong positive correlation with ejaculate volume (P = 0.045), viscosity (P < 0.001) and sperm total motility (P = 0.049). Furthermore, their concentrations exhibited clear seasonal variations in camel semen. In conclusion, the assessment of proAKAP4 concentrations in camel sperm provides a novel parameter to assess sperm quality. Further studies should be performed to investigate proAKAP4 concentrations relative to fertility in Camelidae that may help to define the right time for mating and semen collection and increase the success of breeding programs.
Lay summary
Breeding related to the seasons/time of year in the camel has been reported in several studies. A better knowledge of semen quality during the breeding season would assist in determining the best period for mating in camels. However, conventional sperm parameters are held to be unsatisfactory because they cannot predict breeding potential. ProAKAP4 a sperm-specific protein has been described as a functional marker of sperm and a key fertility marker in several species but has not been described in camels. Motility or membrane integrity parameters of semen collected throughout the breeding season and also the presence of proAKAP4 protein were investigated. ProAKAP4 was identified for the first time in camels and their concentrations exhibited clear seasonal variations in camel semen showing strong correlations with ejaculate volume and total motility and viscosity. Further studies should be performed to investigate proAKAP4 concentrations relative to fertility in camels to define the right time for mating and increase the success of breeding programs.
Jenna Lowe and Erin Curry
Previous reports indicate that red pandas (Ailurus fulgens styani) may experience fetal loss during gestation; however, neither the rate nor timing of pregnancy failure has been described in this species. The objective of this study was to utilize ultrasound video and images collected between 2010 and 2020 at the Cincinnati Zoo and Botanical Garden to better characterize pregnancy loss and fetal development. Trans-abdominal ultrasound examinations were performed on six female red pandas over a 10-year period, resulting in 12 profiles. Pregnancy was diagnosed via ultrasound in 10 of 12 profiles, and 40.0% of pregnancies showed evidence of fetal loss prior to parturition. Pregnancy loss was classified into lost (2 of 10; 20.0%), in which no cubs were produced, or partial loss (2 of 10; 20.0%), in which two concepti were visualized via ultrasound, but only one cub was born. Fetal loss occurred between days 51 and 23 pre-partum. Fetal growth characteristics were documented, including skeletal ossification (occurring between days 32 and 27 pre-partum), crown-rump length, head length, cranial length, and fetal heart rate (173–206 b.p.m.). These findings provide novel insights into pregnancy loss, may serve as a reference for milestones of fetal development, and may be useful in diagnosing pregnancy and assessing pregnancy loss in red pandas.
Lay summary
For many wildlife species, there is no non-invasive method of determining pregnancy; therefore, the rate of pregnancy loss oftentimes is unknown. Many red pandas in human care that are paired for breeding are observed exhibiting normal mating behaviors; however, only a relatively low proportion of females produce cubs. We utilized animals conditioned for ultrasound examination to diagnose pregnancy and characterize the incidence and timing of pregnancy loss. In total, 12 potential pregnancies were monitored, beginning after breeding season and ending ~2 weeks prior to anticipated cubbing. Of these, ten were (83.3%) were diagnosed as pregnant, with 40% undergoing either full or partial pregnancy loss. Fetal growth characteristics, such as body length and head size, are described which may be useful for monitoring pregnancies and estimating fetal age. Results of this study provide novel data on pregnancy loss in red pandas. Insights into the rate and timing of reproductive failure may illuminate causes and contributing factors, ultimately allowing for improvements in husbandry which may result in greater reproductive success of individuals recommended for breeding.
C C Repelaer van Driel-Delprat, E W C M van Dam, P M van de Ven, K Aissa, M K ter Haar, Y Feenstra, A de Roos, G Beelen, R Schats, and C B Lambalk
Graphical Abstract
Abstract
Studies evaluating pregnancy outcomes after assisted reproductive treatment (ART) in women with high-normal (2.5–4.5 mIU/L) thyroid-stimulating hormone (TSH) levels are conflicting, possibly due to different patient charactistics and subfertility indications. The aim of this study was to examine the hypothesis that high-normal compared to low-normal TSH levels are associated with adverse implications for pregnancy outcomes in conventional in vitro fertilization (IVF)-treated women. Therefore, we analyzed retrospectively the characteristics and pregnancy outcomes of 949 subfertile women with TSH 0.3–4.5 mIU/L, treated with conventional IVF between January 2008 and March 2012. Demographic and baseline characteristics were compared between groups of patients based on TSH quartiles, using one-way Anova, Kruskal–Wallis ANOVA and chi-square test. Women with high-normal quartile TSH were significantly more likely to be primary subfertile (P = 0.01), with a higher prevalence of unexplained subfertility and with 15% fewer live births after IVF compared to lower TSH quartiles (P = 0.02). In secondary subfertile women with high-normal TSH, male factor subfertility prevailed (P = 0.01), with more live births (P = 0.01). When analyzing primary and secondary subfertile women as one group, these differences failed to be observed, showing no differences in cumulative pregnancy outcomes of IVF between TSH quartiles (I: 0.3–1.21 mIU/L; II: 1.22–1.68 mIU/L; III: 1.69–2.31 mIU/L; IV: 2.32–4.5 mIU/L). In conclusion, primary subfertile women predominate in the high-normal TSH quartile, associated with significantly fewer live births in a subgroup of primary unexplained subfertile women (9%; n = 87/949), while in secondary subfertile women, dominated by male factor subfertility, high-normal TSH is associated with more live births.
Lay summary
Thyroid hormones are required for all cell processes in the body. An underactive thyroid gland, in which insufficient thyroid hormones are produced and thyroid-stimulating hormone (TSH) rises, is associated with a lower chance of pregnancy. It is not yet clear above which TSH level, 4.5 or also 2.5 mIU/L, this lower probability occurs. Therefore, in 949 couples treated with conventional IVF, we examined whether high-normal TSH levels (TSH: 2.5–4.5 mIU/L) compared to low normal TSH levels (0.3–2.5 mIU/L) affect the live birth rate. We found that women who were trying to become pregnant for the first time, especially without any other cause, that is unexplained subfertility, were more likely to have higher TSH levels. These women had a much lower chance of having a baby compared to women with low-normal TSH levels.
Rachel T Cox, Joanna Poulton, and Suzannah A Williams
There is a worldwide trend for women to have their first pregnancy later in life. However, as oocyte quality declines with maternal aging, this trend leads to an increase in subfertility. The cellular mechanisms underlying this decline in oocyte competence are poorly understood. Oocyte mitochondria are the subcellular organelles that supply the energy that drives early embryogenesis, and thus their quality is critical for successful conception. Mitochondria contain their own DNA (mtDNA) and mutations in mtDNA cause mitochondrial diseases with severe symptoms, such as neurodegeneration and heart disease. Since mitochondrial function declines in tissues as humans age accompanied by an accumulation of mtDNA mutations, mtDNA is implicated as a cause of declining oocyte quality in older mothers. While this mutation load could be caused by declining accuracy of the mitochondrial replisome, age-related decline in mitochondrial quality control likely contributes, however knowledge is lacking. Mitophagy, a cellular process which specifically targets and recycles damaged mitochondria may be involved, but studies are scarce. And although assisted reproductive technologies can help older mothers, how these techniques affect the mechanisms that regulate mitochondrial and oocyte quality have not been studied. With the long-term goal of understanding the molecular mechanisms that control mitochondrial quality in the oocyte, model systems including Drosophila and mouse as well as human oocytes have been used. In this review, we explore the contribution of mitophagy to oocyte quality and the need for further systematic investigation in oocytes during maternal aging using different systems.
Lay summary
Mitochondria are small parts of cells called organelles that generate the chemical energy needed for life. Hundreds of thousands of mitochondria in the developing eggs of the mother support the initial growth and development of the fertilized egg. However, due to increasingly diminished function over time, mitochondria generate less energy as we age, posing real problems for older women considering pregnancy. It is possible that this declining energy could be responsible for declining fertility as women age. Energy may decline because mitochondria fail and the cell’s way of keeping them healthy become less efficient as we age. This review summarizes what is known about mitochondrial quality control in developing eggs as they age. In the future, understanding how the best mitochondria are selected and maintained in the egg, and hence the future baby, may enable older women with or without mitochondrial problems, to have healthy children.
Sharon R Ladyman, Caroline M Larsen, Rennae S Taylor, David R Grattan, and Lesley M E McCowan
Prolactin and placental lactogens increase during pregnancy and are involved with many aspects of maternal metabolic adaptation to pregnancy, likely to impact on fetal growth. The aim of this study was to determine whether maternal plasma prolactin or placental lactogen concentrations at 20 weeks of gestation were associated with later birth of small-for-gestational-age babies (SGA). In a nested case–control study, prolactin and placental lactogen in plasma samples obtained at 20 weeks of gestation were compared between 40 women who gave birth to SGA babies and 40 women with uncomplicated pregnancies and size appropriate-for-gestation-age (AGA) babies. Samples were collected as part of the 'screening of pregnancy endpoints' (SCOPE) prospective cohort study. SGA was defined as birthweight <10th customized birthweight centile (adjusted for maternal weight, height, ethnicity, parity, infant sex, and gestation age) in mothers who remained normotensive. No significant differences were observed in concentrations of prolactin or placental lactogen from women who gave birth to SGA babies compared with women with uncomplicated pregnancies. However, a sex-specific association was observed in SGA pregnancies, whereby lower maternal prolactin concentration at 20 weeks of gestation was observed in SGA pregnancies that were carrying a male fetus (132.0 ± 46.7 ng/mL vs 103.5 ± 38.3 ng/mL, mean ± s.d., P = 0.036 Student’s t-test) compared to control pregnancies carrying a male fetus. Despite the implications of these lactogenic hormones in maternal metabolism, single measurements of either prolactin or placental lactogen at 20 weeks of gestation are unlikely to be useful biomarkers for SGA pregnancies.
Lay summary
Early identification during pregnancy of small for gestational age (SGA) babies would enable interventions to lower risk of complications around birth (perinatal), but current detection rates of these at risk babies is low. Pregnancy hormones, prolactin and placental lactogen, are involved in metabolic changes that are required for the mother to support optimal growth and development of her offspring during pregnancy. The levels of these hormones may provide a measurable indicator (biomarker) to help identify these at risk pregnancies. Levels of these hormones were measured in samples from week 20 of gestation from women who went on to have SGA babies and control pregnancies where babies were born at a size appropriate for gestation age. Despite the implications of prolactin and placental lactogen in maternal metabolism, no significant differences were detected suggesting that single measures of either prolactin or placental lactogen at 20 weeks gestation are unlikely to be useful biomarker to help detect SGA pregnancies.
Gabriel Maicas, Mathew Leonardi, Jodie Avery, Catrina Panuccio, Gustavo Carneiro, M Louise Hull, and George Condous
Objectives
Pouch of Douglas (POD) obliteration is a severe consequence of inflammation in the pelvis, often seen in patients with endometriosis. The sliding sign is a dynamic transvaginal ultrasound (TVS) test that can diagnose POD obliteration. We aimed to develop a deep learning (DL) model to automatically classify the state of the POD using recorded videos depicting the sliding sign test.
Methods
Two expert sonologists performed, interpreted, and recorded videos of consecutive patients from September 2018 to April 2020. The sliding sign was classified as positive (i.e. normal) or negative (i.e. abnormal; POD obliteration). A DL model based on a temporal residual network was prospectively trained with a dataset of TVS videos. The model was tested on an independent test set and its diagnostic accuracy including area under the receiver operating characteristic curve (AUC), accuracy, sensitivity, specificity, and positive and negative predictive value (PPV/NPV) was compared to the reference standard sonologist classification (positive or negative sliding sign).
Results
In a dataset consisting of 749 videos, a positive sliding sign was depicted in 646 (86.2%) videos, whereas 103 (13.8%) videos depicted a negative sliding sign. The dataset was split into training (414 videos), validation (139), and testing (196) maintaining similar positive/negative proportions. When applied to the test dataset using a threshold of 0.9, the model achieved: AUC 96.5% (95% CI: 90.8–100.0%), an accuracy of 88.8% (95% CI: 83.5–92.8%), sensitivity of 88.6% (95% CI: 83.0–92.9%), specificity of 90.0% (95% CI: 68.3–98.8%), a PPV of 98.7% (95% CI: 95.4–99.7%), and an NPV of 47.7% (95% CI: 36.8–58.2%).
Conclusions
We have developed an accurate DL model for the prediction of the TVS-based sliding sign classification.
Lay summary
Endometriosis is a disease that affects females. It can cause very severe scarring inside the body, especially in the pelvis − called the pouch of Douglas (POD). An ultrasound test called the 'sliding sign' can diagnose POD scarring. In our study, we provided input to a computer on how to interpret the sliding sign and determine whether there was POD scarring or not. This is a type of artificial intelligence called deep learning (DL). For this purpose, two expert ultrasound specialists recorded 749 videos of the sliding sign. Most of them (646) were normal and 103 showed POD scarring. In order for the computer to interpret, both normal and abnormal videos were required. After providing the necessary inputs to the computer, the DL model was very accurate (almost nine out of every ten videos was correctly determined by the DL model). In conclusion, we have developed an artificial intelligence that can interpret ultrasound videos of the sliding sign that show POD scarring that is almost as accurate as the ultrasound specialists. We believe this could help increase the knowledge on POD scarring in people with endometriosis.
Samara Silva Souza, Francisco Leo Nascimento Aguiar, Benner Geraldo Alves, Kele Amaral Alves, Fabiana Aparecida Santilli Brandão, Danielle Cristina Calado Brito, Ramon da Silva Raposo, Melba Oliveira Gastal, Ana Paula Ribeiro Rodrigues, José Ricardo Figueiredo, Dárcio Ítalo Alves Teixeira, and Eduardo Leite Gastal
Ovarian tissue transplantation methods using cooled and cryopreserved samples have been attractive options for fertility preservation in animal models and humans. The aim of this study was to evaluate the impact of previous exposure to cooling, cryopreservation, and VEGF on the overall efficiency of equine ovarian tissue after heterotopic xenotransplantation in mice. The end points evaluated were follicular morphology and development, follicular and stromal cell densities, angiogenesis (i.e. the density of new and mature blood vessels), collagen types I and III fiber densities, and total fibrosis. Ovaries of adult mares were harvested after ovariectomy, and ovarian fragments were xenografted in the i.p. wall of BALB nude mice. Ten types of treatments involving different combinations of cooling, cryopreservation, xenografting procedures, and VEGF exposure were compared. The novel aspect of this study was the use of equine ovarian tissue xenotransplantation in mice, challenging the fragments with different combinations of treatments. The main findings were (i) cooling but not cryopreservation was effective in preserving the follicular morphology, (ii) a greater percentage of developing follicles but lower follicular and stromal cell densities were observed after ovarian tissue engraftment, (iii) exposure to VEGF increased new and mature vessels in cryopreserved-transplanted tissue, and (iv) an appropriate balance in the collagen types I and III fiber ratio in cooling-transplanted tissue was observed after exposure to VEGF. This study contributes to advancing knowledge in the preservation of ovarian tissue after cooling-cryopreservation and transplantation aiming to be applied to genetically superior/valuable horses, livestock, endangered animals, and, possibly, humans.
Lay summary
Due to ethical limitations involving humans, the female horse (mare) has recently emerged as an alternative model for reproductive comparisons with women to optimize fertility restoration using ovarian tissue transplantation techniques. This study determined if ovarian tissue from donor mares (n = 3), exposed or not to vascular endothelial growth factor (VEGF) before transplantation, better survives for 7 days after transplantation into mouse hosts (n = 12). Tissues submitted to different combinations of cooling, freezing, and transplanting treatments, along with control groups, were evaluated using the parameters morphology, development, the density of immature eggs (follicles), the density of supportive (stromal) cells, collagen protein proportions, and density of blood vessels. Frozen-thawed treatments had lower percentages of normal follicles. Exposure to VEGF increased blood vessel densities in frozen tissue and favored adequate collagen levels in cooled-transplanted treatments. In conclusion, VEGF exposure seems to be beneficial for mare ovarian tissue transplantation and warrants further investigation.
Peter Thiel, Matthew J Burke, Philippa Bridge-Cook, and Mathew Leonardi
The current approach to treating endometriosis is often inadequate or intolerable for many patients. Until more effective therapies are available, we should aim to maximize the effectiveness of our current options. Optimization may be possible by reducing nocebo effects, which are the negative therapeutic effects not directly caused by a treatment. Awareness of these effects, how they arise, and the factors influencing them, is invaluable if we aim to limit their magnitude. The unique nature of endometriosis diagnosis and management is especially prone to nocebo effects due to multiple factors, including diagnostic delays, feelings of invalidation, social transmission of expectations, and persistent symptoms despite numerous treatments. This commentary discusses the origins of these effects in people with endometriosis, methods of limiting nocebo effects, and future research directions.
Lay summary
The term ‘nocebo’ describes the undesirable effects of a medication or treatment that patients may experience which are not directly caused by the treatment (e.g. tiredness from a sugar pill). These arise from pre-existing expectations toward a treatment and are influenced by multiple external factors, including past experiences, online media, personal beliefs, and personality factors. Endometriosis is a disease characterized by cells like those from the inside of the uterus growing outside of the uterus. The complex nature of endometriosis diagnosis and management creates an environment where nocebo effects may affect treatment outcomes. We may be able to limit nocebo effects through awareness and simple actions that strengthen patient–doctor relationships. Effective therapeutic relationships with doctors are crucial in limiting negative expectations and are established through empathy, honesty, and support. Therapeutic relationships built on trust may allow healthcare providers to address negative expectations, nocebo effects, and the misinformation affecting endometriosis management.