collection-human-follicular-fluid-follicle-somatic-cells-immature

Collection of Human Follicular Fluid, Follicle Somatic Cells, and Immature Oocytes from Individuals Undergoing<em> In Vitro </em>Fertilization

Human ovary follicular contents, including follicular fluid (FF), oocytes, cumulus cells, and somatic cells, offer a powerful yet underutilized ...

Northwestern University

Cancer survivorship rates have drastically increased due to improved efficacy of oncologic treatments. Consequently, clinical concerns have shifted from solely focusing on survival to quality of life, with fertility preservation as an important consideration. Among fertility preservation strategies for female patients, ovarian tissue cryopreservation and subsequent reimplantation has been the only clinical option available to cancer survivors with cryopreserved tissue. However, follicle atresia after transplantation and risk of reintroducing malignant cells have prevented this procedure from becoming widely adopted in clinics. Herein, we investigated the encapsulation of ovarian follicles in alginate hydrogels that isolate the graft from the host, yet allows for maturation after transplantation at a heterotopic (i.e., subcutaneous) site, a process we termed in vivo follicle maturation. Survival of multiple follicle populations was confirmed via histology, with the notable development of the antral follicles. Collected oocytes (63%) exhibited polar body extrusion and were fertilized by intracytoplasmic sperm injection and standard in vitro fertilization procedures. Successfully fertilized oocytes developed to the pronucleus (14%), two-cell (36%), and four-cell (7%) stages. Furthermore, ovarian follicles cotransplanted with metastatic breast cancer cells within the hydrogels allowed for retrieval of the follicles, and no mice developed tumors after removal of the implant, confirming that the hydrogel prevented seeding of disease within the host. Collectively, these findings demonstrate a viable option for safe use of potentially cancer-laden ovarian donor tissue for in vivo follicle maturation within a retrievable hydrogel and subsequent oocyte collection. Ultimately, this technology may provide novel options to preserve fertility for young female patients with cancer.

Retrievable hydrogels for ovarian follicle transplantation and oocyte collection.

Upon fertilization or parthenogenesis, zinc is released into the extracellular space through a series of exocytic events termed zinc sparks, which are tightly coordinated with intracellular calcium transients. The zinc spark reduces the total amount of intracellular zinc, and this reduction is necessary and sufficient to induce egg activation even in the absence of calcium transients. In addition, this zinc release contributes to the block to polyspermy through modification of the zona pellucida. The zinc spark has been documented in all organisms examined to date including the mouse, two species of nonhuman primates, and human. Here we determined whether zinc sparks occur in the bovine, an important model of gamete development in mono-ovulatory mammalian species. We obtained metaphase II-arrested (MII) bovine eggs following in vitro maturation. Total zinc, assessed in single cells using X-Ray Fluorescence Microscopy, was significantly more abundant in the bovine egg compared to iron and copper. Studies with intracellular fluorescent probes revealed that labile zinc pools are localized to discrete cytoplasmic punctae enriched at the cortex. To determine whether zinc undergoes dynamic fluxes during egg activation, we parthenogenetically activated bovine eggs using two approaches: ionomycin or bovine phospholipase C zeta (bPlcζ). Both these methods induced zinc sparks coordinately with intracellular calcium transients. The zinc spark was also observed in bovine eggs following intracytoplasmic sperm injection. These results establish that zinc is the most abundant transition metal in the bovine egg, and zinc flux during egg activation - induced by chemical activation or sperm - is a highly conserved event across mammalian species.

Bovine eggs release zinc in response to parthenogenetic and sperm-induced egg activation.

 It is unclear whether a history of thyroid cancer is associated with an increased risk of adverse pregnancy outcomes in subsequent pregnancies. This study aimed to evaluate the risk of adverse obstetric outcomes and the abnormal growth of offspring in women with a history of thyroid cancer.  This retrospective observational study used nationwide data from between 2006 and 2014 to compare pregnancy outcomes of women with a history of thyroid cancer and those with no such history. Cases of thyroid cancer were identified using ICD-10 codes.  During the study period, 7232 women with a history of thyroid cancer and 2,269,051 women without a history of thyroid cancer gave birth. The risks of cesarean section, preterm birth, low birth weight, large for gestational age, preeclampsia, placental abruption, placenta previa, and stillbirth were not different between the groups. Women with a history of thyroid cancer had a statistically higher risk of postpartum hemorrhage (odds ratio [OR] = 1.23 [confidence interval (CI) 1.15-1.32],  < 0.05, corrected with the false discovery rate). Additionally, generalized estimating equations analysis showed that there was no difference in the risk of underweight (OR = 1.05 [CI 0.93-1.19]) and obese (OR = 0.94 [CI 0.84-1.05]) offspring assessed over a period of 80 months after adjusting for confounding factors.  Women with a history of thyroid cancer have similar pregnancy outcomes and offspring growth to those with no such history.

Risk of Adverse Obstetric Outcomes and the Abnormal Growth of Offspring in Women with a History of Thyroid Cancer.

The aim of this study was to investigate how type I diabetes mellitus (T1D) affects the folliculogenesis and oocyte development, fertilization, and embryo development.

Poorly-Controlled Type 1 Diabetes Mellitus Impairs LH-LHCGR Signaling in the Ovaries and Decreases Female Fertility in Mice.

Zinc dynamics are essential for oocyte meiotic maturation, egg activation, and preimplantation embryo development. During fertilisation and egg activation, the egg releases billions of zinc atoms (Zn2+) in an exocytotic event termed the 'zinc spark'. We hypothesised that this zinc transport and exocytosis is dependent upon the intracellular trafficking of cortical granules (CG) which requires myosin-actin-dependent motors. Treatment of mature mouse and human eggs with ML-7, a myosin light chain kinase inhibitor (MLCK), resulted in an 80% reduction in zinc spark intensity compared to untreated controls when activated with ionomycin. Moreover, CG migration towards the plasma membrane was significantly decreased in ML-7-treated eggs compared with controls when activated parthenogenetically with ionomycin. In sperm-induced fertilisation via intracytoplasmic sperm injection (ICSI), ML-7-treated mouse eggs exhibited decreased labile zinc intensity and cortical CG staining. Collectively, these data demonstrate that ML-7 treatment impairs zinc release from both murine and human eggs after activation, demonstrating that zinc exocytosis requires myosin light chain kinase activity. Further, these results provide additional support that zinc is likely stored and released from CGs. These data underscore the importance of intracellular zinc trafficking as a crucial component of egg maturation necessary for egg activation and early embryo development.

Zinc exocytosis is sensitive to myosin light chain kinase inhibition in mouse and human eggs.

Poor oral health is not only associated with diabetes and cardiovascular disease but adverse pregnancy outcomes. However the influence of dental caries on pregnancy is unknown. The aim of this study was to evaluate the association between dental caries and adverse pregnancy outcomes and the effect of treatment for dental caries on adverse pregnancy outcomes. Primiparas who delivered a singleton between January 1, 2010 and December 31, 2014 and underwent both general health examination and oral health examination during a National Korea Health Screening Examination within 1 year of pregnancy were eligible. The data of the women who met the inclusion criteria were linked to the data of their offspring contained within the National Korea Health Screening Program for Infants and Children database. Among 120,622 women who delivered during the study period, 28,623 (23.7%) women had dental caries. Among them, 4,741 (16.6%) women were treated for dental caries after diagnosis. In a multivariable analysis, women with dental caries had an increased risk of delivering large-for-gestational-age infants (odds ratio, 1.15; 95% confidence interval, 1.07, 1.23) compared to those without dental caries. When women with dental caries were divided on the basis of the treatment of dental caries, women with dental caries but no treatment had an increased risk of delivering large-for-gestational-age infants (odds ratio, 1.15; 95% confidence interval, 1.06, 1.24); conversely, there was no increased risk in women with dental caries and treatment compared with those without. Dental caries and its treatment were not associated with preterm birth and preeclampsia. Untreated dental caries was not associated with preterm birth or preeclampsia but with the risk of delivering large-for-gestational-age infants. These whole observation may be attributed to the various characteristics of mothers who develop dental caries are not treated.

Association between dental caries and adverse pregnancy outcomes.

Childhood cancer patients undergoing cancer therapy can be rendered infertile during adulthood. With more girls surviving cancer, fertility preservation in young cancer patients is a major clinical challenge. Advances in egg culture may offer benefits for the fertility of these patients in the future.

Prospects of Rescuing Young Eggs for Oncofertility.

Although only one spermatozoon is needed to create a zygote, a significant challenge is the storage and recovery of germ cells when sperm counts are extremely low.

A decellularized oocyte-derived scaffold provides a "sperm safe" to preserve mammalian spermatozoa.

The discovery of PLCZ1 nearly 20 years ago as the primary Ca2+ oscillation-inducing factor in the sperm of mammals represented a significant breakthrough in our quest to elucidate the molecules and pathways that promote egg activation during fertilization. The advent of the intracytoplasmic sperm injection (ICSI) technique, which made fertilization possible without sperm capacitation, acrosome reaction, and gamete fusion, strengthened the research that led to the discovery of PLCZ1 and became an essential clinical tool for humans. The use of ICSI combined with the detection of PLCZ1 expression and mutations in infertile patients established the fundamental role of PLCZ1 in human fertility while leading to the discovery of novel components of the perinuclear theca, the site of the residence of PLCZ1 in sperm before fertilization. Remarkably, the more extensive use of ICSI in species other than humans and mice revealed poor success and exposed gaps in our understanding of PLCZ1 release and/or activation. Similarly, fertilization using sperm from mouse models lacking Plcz1 has produced striking results whose true implications are yet to be determined. Nevertheless, answers to these unresolved questions will produce a complete picture of the adaptations and molecular players that mammalian species employ to ensure the success of the triggering event of embryo development that has linked generations since the beginning of times.

SPERM FACTORS AND EGG ACTIVATION: ICSI and the discovery of the sperm factor and PLCZ1.

Meiotic maturation and cumulus expansion are essential for the generation of a developmentally competent gamete, and both processes can be recapitulated in vitro. We used a closed time-lapse incubator (EmbryoScope+™) to establish morphokinetic parameters of meiotic progression and cumulus expansion in mice and correlated these outcomes with egg ploidy. The average time to germinal vesicle breakdown (GVBD), time to first polar body extrusion (PBE), and duration of meiosis I were 0.91 ± 0.01, 8.82 ± 0.06, and 7.93 ± 0.06 h, respectively. The overall rate of cumulus layer expansion was 0.091 ± 0.002 μm/min, and the velocity of expansion peaked during the first 8 h of in vitro maturation (IVM) and then slowed. IVM of oocytes exposed to Nocodazole, a microtubule disrupting agent, and cumulus oocyte complexes (COCs) to 4-methylumbelliferone, a hyaluronan synthesis inhibitor, resulted in a dose-dependent perturbation of morphokinetics, thereby validating the system. The incidence of euploidy following IVM was &gt;90% for both denuded oocytes and intact COCs. No differences were observed between euploid and aneuploid eggs with respect to time to GVBD (0.90 ± 0.22 vs. 0.97 ± 0.19 h), time to PBE (8.89 ± 0.98 vs. 9.10 ± 1.42 h), duration of meiosis I (8.01 ± 0.91 vs. 8.13 ± 1.38 h), and overall rate and kinetics of cumulus expansion (0.089 ± 0.02 vs 0.088 ± 0.03 μm/min) (P &gt; 0.05). These morphokinetic parameters provide novel quantitative and non-invasive metrics for the evaluation of meiotic maturation and cumulus expansion and will enable screening compounds that modulate these processes.

Quantitative morphokinetic parameters identify novel dynamics of oocyte meiotic maturation and cumulus expansion†.

Morphokinetic analysis using a closed time-lapse monitoring system (EmbryoScope + ™) provides quantitative metrics of meiotic progression and cumulus expansion. The goal of this study was to use a physiologic aging mouse model, in which egg aneuploidy levels increase, to determine whether there are age-dependent differences in morphokinetic parameters of oocyte maturation.

Morphokinetic parameters of mouse oocyte meiotic maturation and cumulus expansion are not affected by reproductive age or ploidy status.

To accurately phenocopy human biology , researchers have been reducing their dependence on standard, static two-dimensional (2D) cultures and instead are moving towards three-dimensional (3D) and/or multicellular culture techniques. While these culture innovations are becoming more commonplace, there is a growing body of research that illustrates the benefits and even necessity of recapitulating the dynamic flow of nutrients, gas, waste exchange and tissue interactions that occur . However, cost and engineering complexity are two main factors that hinder the adoption of these technologies and incorporation into standard laboratory workflows. We developed LATTICE, a plug-and-play microfluidic platform able to house up to eight large tissue or organ models that can be cultured individually or in an interconnected fashion. The functionality of the platform to model both healthy and diseased tissue states was demonstrated using 3D cultures of reproductive tissues including murine ovarian tissues and human fallopian tube explants (hFTE). When exogenously exposed to pathological doses of gonadotropins and androgens to mimic the endocrinology of polycystic ovarian syndrome (PCOS), subsequent ovarian follicle development, hormone production and ovulation copied key features of this endocrinopathy. Further, hFTE cilia beating decreased significantly only when experiencing continuous media exchanges. We were then able to endogenously recreate this phenotype on the platform by dynamically co-culturing the PCOS ovary and hFTE. LATTICE was designed to be customizable with flexibility in 3D culture formats and can serve as a powerful automated tool to enable the study of tissue and cellular dynamics in health and disease in all fields of research.

A new tissue-agnostic microfluidic device to model physiology and disease: the lattice platform.

Is there an association between morphokinetic variables of meiotic maturation and the severity of aneuploidy following  maturation (IVM) in the mouse?

Article	Year
Retrievable hydrogels for ovarian follicle transplantation and oocyte collection. Biotechnology and bioengineering\| PubMed ID: 29704433	2018
Bovine eggs release zinc in response to parthenogenetic and sperm-induced egg activation. Theriogenology\| PubMed ID: 30639695	2019
Risk of Adverse Obstetric Outcomes and the Abnormal Growth of Offspring in Women with a History of Thyroid Cancer. Thyroid : official journal of the American Thyroid Association\| PubMed ID: 30957663	2019
Poorly-Controlled Type 1 Diabetes Mellitus Impairs LH-LHCGR Signaling in the Ovaries and Decreases Female Fertility in Mice. Yonsei medical journal\| PubMed ID: 31250581	2019
Zinc exocytosis is sensitive to myosin light chain kinase inhibition in mouse and human eggs. Molecular human reproduction\| PubMed ID: 32119740	2020
Association between dental caries and adverse pregnancy outcomes. Scientific reports\| PubMed ID: 32210330	2020
Prospects of Rescuing Young Eggs for Oncofertility. Trends in endocrinology and metabolism: TEM\| PubMed ID: 32807599	2020
A decellularized oocyte-derived scaffold provides a "sperm safe" to preserve mammalian spermatozoa. Andrology\| PubMed ID: 33565255	2021
SPERM FACTORS AND EGG ACTIVATION: ICSI and the discovery of the sperm factor and PLCZ1. Reproduction (Cambridge, England)\| PubMed ID: 35521902	2022
Quantitative morphokinetic parameters identify novel dynamics of oocyte meiotic maturation and cumulus expansion†. Biology of reproduction\| PubMed ID: 35810327	2022
Morphokinetic parameters of mouse oocyte meiotic maturation and cumulus expansion are not affected by reproductive age or ploidy status. Journal of assisted reproduction and genetics\| PubMed ID: 37012451	2023
A new tissue-agnostic microfluidic device to model physiology and disease: the lattice platform. Lab on a chip\| PubMed ID: 37846545	2023
The severity of meiotic aneuploidy is associated with altered morphokinetic variables of mouse oocyte maturation. Human reproduction open\| PubMed ID: 38764910	2024

Hoi Chang Lee

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