Reproduction & Fertility最新文献

Purpose Can a comprehensive flow cytometry panel be used to assess immunophenotype profiles in menstrual blood of patients experiencing reproductive failure and age matched controls of proven fertility? Methods 58 recurrent pregnancy loss and repeated implantation failure patients, along with 15 age matched controls of proven fertility, had menstrual blood samples obtained within the first 24 hours of the onset of menstruation to non-invasively assess the local immunophenotype. Using a comprehensive multi-parameter flow panel the lymphocyte sub-populations were described and compared. Results Relative to well established peripheral blood immunophenotyping values, distinct lymphocyte population differences were noted between the subgroups. The ratios of CD4+ and CD8+ T-cells were inverted relative to peripheral blood and uterine NK cells represented by CD56bright were distinctly visualised, emphasising the distinction of menstrual and peripheral blood. Relative to controls there were marked increases in CD3+ve T-cells (p=0.009), CD4:CD8 ratio (p=0.004), CD19 B-cells (p=0.026) and CD56dim NK's (p=0.002) in the reproductive failure cases. Conclusions Flow cytometric evaluation can provide a rapid and objective analysis of lymphocyte subpopulations in many forms of tissue and fluid. The findings show significant variations in cellular composition of immune cells indicating a distinct compartment, with differences between cases and controls. Immunological assessment of the menstrual blood immunophenotype, in clinically appropriate patients, may provide insight into the aetiology of adverse reproductive outcome, without the risks and inconveniences associated with a more invasive endometrial biopsy.

Optimisation of lifestyle factors such as smoking and alcohol are encouraged to improve fecundability rates in the fertility setting. Currently, routine fertility consultations do not involve counselling or imparting advice regarding habitual physical activity (PA) and/or structured exercise, despite data showing that vigorous PA can be associated with delayed time to pregnancy. Therefore, this study aimed to determine habitual PA in a sample of women attending the one stop infertility (OSI) clinic. 250 women attending a large tertiary level NHS fertility unit prospectively anonymously completed a questionnaire over a period of 9 months. Participant's (mean age 34±5years, mean BMI 29±7kg/m2) habitual PA levels varied from vigorous exercise on ≥5 days/week (8%, n=17), to no moderate or high intensity activities across the whole week (66%, n=29). The majority of women reported no structured exercise (72%, n=179). No association was identified between any domain of PA and BMI, age, alcohol units, regular periods, or time spent trying to conceive (P > 0.05). Participant's habitual PA levels varied widely and no association between any domain of PA and background of the women was identified. No existing evidence and/or guidelines to explicitly inform women attempting to conceive regarding recommended PA levels are available, despite PA being a modifiable, affordable, and feasible lifestyle choice with the possible potential to improve fertility. A large-scale, clinical trial assessing effects of PA on fecundability is warranted to gain insights into the potential of this lifestyle factor to improve fertility outcomes and to explore the underlying biological mechanisms involved.

Abstract: During fertilization, avian sperm preferentially penetrate into the perivitelline membrane that covers the germinal disk region where the female nucleus is present. This phenomenon has been observed not only in domestic birds but also in wild birds; however, the mechanisms controlling sperm preference are still unclear. In this study, we investigated the possible involvement of annexin family protein in sperm-egg interaction in Japanese quail. Microscopic examination of fertilized eggs indicated that quail sperm penetration only occurred in the germinal disk region, and sperm localized outside the germinal disk were trapped in the perivitelline membrane. Western blot analysis and immunofluorescence microscopy revealed the presence of annexin A1 and A6 in the oocyte membrane, while annexin A6 localized in the perivitelline space of the germinal disk region. Further, our sperm binding assay using recombinant annexin A6 demonstrated that ejaculated sperm specifically bound to annexin A6 expressed in mammalian cell lines. These results suggest that annexin A6, which is expressed on the surface of oocytes, may function in sperm-egg interaction in the germinal disk region and that this binding may ensure sperm retention on the surface of the egg plasma membrane until fertilization takes place in Japanese quail.

Lay summary: In bird species, fertilization takes place immediately after ovulation of the egg. Sperm preferentially penetrate a specific area of the egg coating that covers the 'germinal disk region' - this area contains the cell that needs to be fertilized by a sperm. However, since the bird egg is extremely large in size and sperm must reach the 'germinal disk region' to achieve fertilization, it is unclear how this happens. Annexin proteins support fertilization in mammals, and we found that annexin A6 protein exhibits a unique localization in the germinal disk region in the eggs of Japanese quail. To test this interaction, we incubated quail sperm with cells that produced annexin A6 and found that ejaculated sperm bound to the cells. These results suggest that annexin A6 may have a role in the sperm-egg interaction in the germinal disk region in Japanese quail.

In vitro: culturing of endometrial cells obtained from the uterine mucosa or ectopic sites is used to study molecular and cellular signalling relevant to physiologic and pathologic reproductive conditions. However, the lack of consensus on standard operating procedures for deriving, characterising and maintaining primary cells in two- or three-dimensional cultures from eutopic or ectopic endometrium may be hindering progress in this area of research. Guidance for unbiased in vitro research methodologies in the field of reproductive science remains essential to increase confidence in the reliability of in vitro models. We present herein the protocol for a Delphi process to develop a consensus on in vitro methodologies using endometrial cells (ENDOCELL-Seud Project). A steering committee composed of leading scientists will select critical methodologies, topics and items that need to be harmonised and that will be included in a survey. An enlarged panel of experts (ENDOCELL-Seud Working Group) will be invited to participate in the survey and provide their ratings to the items to be harmonised. According to Delphi, an iterative investigation method will be adopted. Recommended measures will be finalised by the steering committee. The study received full ethical approval from the Ethical Committee of the Maastricht University (ref. FHML-REC/2021/103). The study findings will be available in both peer-reviewed articles and will also be disseminated to appropriate audiences at relevant conferences.

Lay summary: Patient-derived cells cultured in the lab are simple and cost-effective methods used to study biological and dysfunctional or disease processes. These tools are frequently used in the field of reproductive medicine. However, the lack of clear recommendations and standardised methodology to guide the laboratory work of researchers can produce results that are not always reproducible and sometimes are incorrect. To remedy this situation, we define here a method to ascertain if researchers who routinely culture cells in the lab agree or disagree on the optimal laboratory techniques. This method will be used to make recommendations for future researchers working in the field of reproductive biology to reproducibly culture endometrial cells in the laboratory.

The manuscript has been submitted without altering abstract in line with Reproduction's Flexible Submission Process. The abstract is extended and thus does not fit this space.

Endometrial glands are essential for fertility, consisting of ciliated and secretory cells that facilitate a suitable uterine environment for embryo implantation. This study sought to determine whether an endometrial gland specific transcriptome and splicing profile are altered in women with recurrent pregnancy loss. Our data provide a comprehensive catalogue of cilia and PAEP gene isoforms and relative exon usage in endometrial glands. We report a previously unannotated endometrial gland cilia transcript GALNT11 and its susceptibility to exon skipping. Key endometrial receptivity gene transcripts are also reported to change in endometrial glands of women with recurrent pregnancy loss. The endometrial gland cilia and PAEP targets identified in this study could be used to identify a perturbed endometrium, isolate causes of recurrent pregnancy loss and develop targeted therapies in personalised medicine.

The need to develop new treatments to prevent unprompted premature delivery before 37 weeks of pregnancy remains pressing and unmet. Bacteria (Lactobacillus species) that promote vaginal health produce biochemical compounds that prevent the growth of microbes such as Gardnerella vaginalis. Overgrowth of G. vaginalis can cause vaginal infection with smelly discharge and increase a woman's risk of sexually transmitted infections and premature delivery. In this study, we examined how normal health-promoting (L. crispatus) and potentially harmful (G. vaginalis) vaginal bacteria interact in a laboratory setting. This was in order to observe natural and effective agent(s) from L. crispatus that can hinder the growth of G. vaginalis and accompanying immune response. We observed that L. crispatus clears G. vaginalis by itself and with several biochemical compounds that it produces. Such biochemical compounds can be developed into treatment for vaginal infections and premature delivery due to infection and inappropriate immune response.

The use of intracytoplasmic sperm injection (ICSI) has recently increased worldwide. The live birth rate per ICSI cycle is low, and over half of infertile couples remain childless. Chromosomal polymorphisms are up to five times more common in couples with infertility compared to the general population. We aimed to investigate the association between chromosomal polymorphisms and reproductive outcomes in couples undergoing ICSI treatment. We analysed 942 ICSI fresh and frozen embryo transfer cycles in 697 women who underwent karyotyping analysis using Giemsa-Trypsin-Leishman banding prior to assisted conception at the Fertility Centre of Lanka Hospitals, Sri Lanka, between 2016 and 2018. The primary outcomes were pregnancy, miscarriage, and live birth rates. We compared outcomes according to the presence or absence of chromosomal polymorphism in females, males and couples. There were 294 pregnancies (31.2%) recorded in the study; 130 suffered a miscarriage (13.8%), 13 were ectopic pregnancies (1.3%) and 151 resulted in a live birth (16.0%). The evidence from univariable and multivariable analyses (adjusted for age, BMI, ovarian reserve and treatment type) did not confidently identify a difference in pregnancy, miscarriage or live birth rates between couples with no chromosomal polymorphisms compared to couples where the female, male or both partners were carriers of a chromosomal polymorphism. Further, we did not identify a clear association between the presence of chromosomal polymorphisms and reproductive outcomes compared to participants without chromosomal polymorphisms. Wide CIs precluded the identification of clinically meaningful associations.

Lay summary: Infertility affects approximately one in eight couples worldwide. The use of intracytoplasmic sperm injection (ICSI), where the sperm is directly injected into an egg using a micromanipulator outside the body, has become particularly popular in recent years. However, the success rate remains low. In human cells, the genetic material is arranged in structures called chromosomes. Chromosomal polymorphism is a normal variation where the genetic material is arranged differently to the average individual and is more common in infertile couples compared to the general population. We analysed data from 942 ICSI cycles in 697 couples who underwent karyotyping analysis to assess the changes in chromosomes between 2016 and 2018. The pregnancy rate was 31.2%, with 16.0% of participants experiencing a live birth, while 13.8% of pregnancies resulted in a miscarriage and 1.3% were outside the womb cavity (ectopic). The evidence did not identify a clear association between the chromosomal polymorphism and the outcome of treatment.

Abstract: Giant pandas are mono-estrus seasonal breeders, with the breeding season typically occurring in the spring. Successful fertilization is followed by an embryonic diapause, of variable length, with birth in the late summer/autumn. There is a need for additional understanding of giant panda reproductive physiology, and the development of enhanced biomarkers for impending proestrus and peak fertility. We aimed to determine the utility of non-invasive androgen measurements in the detection of both proestrus and estrus. Urine from 20 cycles (-40 days to +10 days from peak estrus) from 5 female giant pandas was analyzed for estrogen, progestogens and androgens (via testosterone and DHEA assays), and hormone concentrations were corrected against urinary specific gravity. Across proestrus, estrogens increased while progestogens and androgens decreased - at the point of entry into proestrus, androgens (as detected by the testosterone assay) decreased prior to progestogens and gave 4 days advanced warning of proestrus. At the time of peak estrus, androgens (as detected by the DHEA assay) were significantly increased at the time of the decrease in estrogen metabolites from the peak, acting as an alternative confirmatory indicator of the fertile window. This novel finding allows for enlargement of the preparative window for captive breeding and facilitates panda management within breeding programmes. Androgens allow an enhanced monitoring of giant panda estrus, not only advancing the warning of impending proestrus, but also prospectively identifying peak fertility.

Lay summary: Giant pandas have one chance at pregnancy per year. The 2-day fertile window timing varies by year and panda. This is monitored by measuring the level of estrogens in the urine, which increase, indicating an upcoming fertile period. After 1-2 weeks of increase, estrogens peak and fall, marking the optimal fertile time. We tested other hormones to see if we can predict the fertile window in advance, and the specific fertile time with more accuracy. In 20 breeding seasons from 5 females, we found androgens, usually thought of as male hormones, had an important role. Testosterone gives 4 days advanced warning of estrogens increasing. DHEA identified peak estrogen and the fertile time before needing to see a confirmed decrease in estrogen itself. Therefore, androgens help improve monitoring of the giant panda breeding season, giving early warning of fertility, key in facilitating captive breeding and giant panda conservation.