New Insights into the Mammalian Egg Zona Pellucida.

Mammalian oocytes are surrounded by an extracellular coat called the zona pellucida (ZP), which, from an evolutionary point of view, is the most ancient of the coats that envelope vertebrate oocytes and conceptuses. This matrix separates the oocyte from cumulus cells and is responsible for species-specific recognition between gametes, preventing polyspermy and protecting the preimplantation embryo. The ZP is a dynamic structure that shows different properties before and after fertilization. Until very recently, mammalian ZP was believed to be composed of only three glycoproteins, ZP1, ZP2 and ZP3, as first described in mouse. However, studies have revealed that this composition is not necessarily applicable to other mammals. Such differences can be explained by an analysis of the molecular evolution of the ZP gene family, during which ZP genes have suffered pseudogenization and duplication events that have resulted in differing models of ZP protein composition. The many discoveries made in recent years related to ZP composition and evolution suggest that a compilation would be useful. Moreover, this review analyses ZP biosynthesis, the role of each ZP protein in different mammalian species and how these proteins may interact among themselves and with other proteins present in the oviductal lumen.

Embryological Characteristics and Clinical Outcomes of Oocytes with Heterogeneous Zona Pellucida During Assisted Reproduction Treatment: A Retrospective Study.

BACKGROUND The condition of the zona pellucida can be used to predict human oocyte quality. This study investigated the embryological characteristics and clinical outcomes of oocytes with heterogeneous zona pellucida (HZP) during in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI). MATERIAL AND METHODS This was a retrospective study of IVF and ICSI cycles undertaken at The First Affiliated Hospital of Wenzhou Medical University between June 2006 and March 2016. Cycles involving oocytes with HZP (HZP group) were compared with those involving non-HZP oocytes retrieved on the same day (non-HZP group). Embryological characteristics and clinical outcomes were compared. RESULTS There were 29 IVF and 46 ICSI cycles in the HZP group, and 521 IVF and 206 ICSI cycles in the non-HZP group. In ICSI cycles, the rates of MII oocyte and high-quality embryo were lower in the HZP group (p<0.05 vs. non-HZP). In IVF cycles, the MII oocyte (p<0.001), normal fertilization (p<0.001), and cleavage (p<0.001) rates were lower, while the abandoned transfer rate (p<0.001) was higher in the HZP group compared with the non-HZP group. The positive human chorionic gonadotropin (HCG), implantation, pregnancy, and miscarriage rates were similar between groups. Multivariate analysis revealed that the woman's age (OR=0.916 95% CI 0.873-0.962; p<0.001) and the number of D3 high-quality embryos (OR=1.120 95% CI 1.004-1.249; p=0.043) were associated with pregnancy in IVF cycles, but no significant factors were found in ICSI cycles. CONCLUSIONS ICSI may help increase the number of viable embryos in cycles with oocytes showing HZP. However, both IVF and ICSI cycles can achieve pregnancy.

Zona Pellucida Proteins, Fibrils, and Matrix.

The zona pellucida (ZP) is an extracellular matrix that surrounds all mammalian oocytes, eggs, and early embryos and plays vital roles during oogenesis, fertilization, and preimplantation development. The ZP is composed of three or four glycosylated proteins, ZP1-4, that are synthesized, processed, secreted, and assembled into long, cross-linked fibrils by growing oocytes. ZP proteins have an immunoglobulin-like three-dimensional structure and a ZP domain that consists of two subdomains, ZP-N and ZP-C, with ZP-N of ZP2 and ZP3 required for fibril assembly. A ZP2-ZP3 dimer is located periodically along ZP fibrils that are cross-linked by ZP1, a protein with a proline-rich N terminus. Fibrils in the inner and outer regions of the ZP are oriented perpendicular and parallel to the oolemma, respectively, giving the ZP a multilayered appearance. Upon fertilization of eggs, modification of ZP2 and ZP3 results in changes in the ZP's physical and biological properties that have important consequences. Certain structural features of ZP proteins suggest that they may be amyloid-like proteins.

Effects of glucuronic acid and N-acetyl-D-glucosamine supplementation on the perivitelline space during the IVM of pig oocytes.

The objective of this study was to minimise polyspermic penetration by increasing the perivitelline space (PVS) thickness through supplementation of the hyaluronic acid components glucuronic acid and N-acetyl-d-glucosamine (GlcNAc). Oocytes (n=4690) were supplemented during the first 24h and/or the remainder of maturation (final 16-18h) with 0.01mM glucuronic acid and 0.01mM GlcNAc and then evaluated for PVS thickness, hyaluronic acid, glutathione and glutathione peroxidase concentrations. Fertilised oocytes were evaluated for polyspermic penetration and embryo development. The PVS thickness and amount of hyaluronic acid was significantly (P<0.05) greater in oocytes supplemented with 0.01mM glucuronic acid and 0.01mM GlcNAc during the second part or all of maturation compared with the other treatments. In addition, polyspermic penetration was significantly (P<0.05) less in oocytes supplemented with 0.01mM glucuronic acid and 0.01mM GlcNAc during the second part or all of maturation compared with the other treatments. Supplementing 0.01mM glucuronic acid and GlcNAc during maturation significantly (P<0.05) increased the percentage of cleaved embryos by 48h after IVF and blastocysts formed by 144h after IVF compared those not supplemented. These results indicate that supplementing PVS components during maturation decreases polyspermic penetration by increasing PVS thickness.

Removing the zona pellucida can decrease cytoplasmic fragmentations in human embryos: a pilot study using 3PN embryos and time-lapse cinematography.

PURPOSE: The aim of this study was to establish a new method of decreasing cytoplasmic fragmentation in early-stage human embryos. METHODS: The zona pellucida (ZP) of abnormally-fertilized oocytes (zygotes with three pronuclei (3PN)), which were donated by patients, was removed at the pronuclear stage. ZP-free embryos were observed in a time-lapse imaging and culturing system in order to examine developmental morphology and embryonic quality. RESULTS: Based on a modification of Veeck's criteria, 47 of 69 ZP-free 3PN embryos (68.1%) showed fragmentation of less than 20% of the total volume of cytoplasm at the first cleavage (grades 1 and 2), 17 (24.6%) showed 20-40% cytoplasmic fragments (grade 3), and only 5 (7.2%) showed more than 40% fragments (grade 4). These results suggest that the rate of fragmentation is decreased by ZP removal before the first cleavage, compared with normal (ZP-intact) 3PN and 2-pronuclear/2-polar body embryos. CONCLUSIONS: This study revealed that the ZP is not always necessary for normal development after the pronuclear stage because the ZP-free embryos studied herein developed normally, maintained their cell adhesion well, and showed a decreased rate of fragmentation. This innovative culture system might provide the major breakthrough needed for patients who have difficulty obtaining good-quality embryos.

Novel ultrastructural findings in bovine oocytes matured in vitro.

The ultrastructural morphology of the bovine oocyte at different maturation stages has been previously analyzed but without detailed structural observations at the mature stage. The objective of the present study was thus to establish the ultrastructural characteristics of the mature bovine oocyte in full detail. Oocytes from Bos taurus (Holstein-Friesian) cows were aspirated from ovaries collected after being slaughtered at a local abattoir. After in vitro culture for 24 h, some of them were processed for electron microscopy. We described the ultrastructure of the zona pellucida, which presented three different regions, and novel cytoplasmic findings. There were two types of electron-lucent vesicles (heterogeneous and striated), which were suggested to give rise to lipid droplets, and presence of receptor-mediated endocytosis. In conclusion, our results indicate that although the mature bovine oocyte is devoid of evident yolk, it might be filled with an extensive lipid factory. In addition, even before fertilization, the mature oocyte seemed to absorb nutrients through receptor-mediated endocytosis, indicating active energy use or storage.

Time-dependent effects of heat shock on the zona pellucida ultrastructure and in vitro developmental competence of bovine oocytes.

The aim of this study was to determine the effects of different exposure lenght to heat shock (HS) during in vitro maturation (IVM) on zona pellucida (ZP) ultrastructure and developmental competence of bovine oocytes. Cumulus-oocyte complexes (COCs) were matured in vitro (IVM) at 38.5 °C for 24 h (control group, CG), or incubated at 41 °C (HS) for 6 h (HS-6h), 12 h (HS-12h), 18 h (HS-18h), and 22h (HS-22h) followed by incubation at 38.5 °C to complete a full 24-h period of maturation. After IVM, oocytes were subjected to scanning electron microscopy (SEM) or in vitro fertilization and culture until the blastocyst stage. For heat-shocked oocytes, with exception of those in the HS-6h group, SEM examinations revealed that ZP surfaces were rough and characterized by a presence of spongy network. Oocytes from the HS-22h group displayed an increase in the number of pores, as well as a higher proportion of oocytes with amorphous ZPs. The proportion of oocytes that reached metaphase II (MII) stage decreased in all HS groups, regardless of the duration of exposure to 41 °C. These results provide evidence that HS during IVM for 12-22 h reduces the developmental competence of bovine oocytes, increasing the percentage of oocytes with abnormal chromosomal organization, and reducing fertilization and blastocysts formation rate. The effects of HS were more pronounced for the 22-h exposure group. The damage induced by HS on oocyte function clearly increased upon exposure to elevated temperature.

Ultrastructural Imaging Analysis of the Zona Pellucida Surface in Bovine Oocytes.

The aims of the present study were to: (i) evaluate the ultrastructural differences in the zona pellucida (ZP) surface between immature and mature bovine oocytes, and (ii) describe a new objective technique to measure the pores in the outer ZP. Intact cumulus-oocyte complexes (COCs) obtained from a local abattoir were immediately fixed (immature group) or submitted to in vitro maturation (IVM) at 38.5 °C for 24 h in a humidified atmosphere of 5% CO2 in air (mature group). Oocytes from both groups were morphologically evaluated via Scanning Electron Microscopy (SEM) and the images were processed in the Fiji/ImageJ software using a new objective methodology through the Trainable Weka Segmentation plugin. The average number of pores in ZP was greater (p 0.05) between groups. In conclusion, it has been shown that the number of pores highlighted the main ultrastructural change in the morphology of the ZP surface of bovine oocytes during the IVM process. We have described an objective method that can be used to evaluate ultrastructural modifications of the ZP surface during oocyte maturation and early embryo development.

Ultrastructural identification of CD9 positive extracellular vesicles released from human embryos and transported through the zona pellucida.

Extracellular vesicles (EVs) are highly specific and multi-purpose vesicular structures that are released by various cell and tissue types in the body. However, the secretion of EVs from mammalian embryos, especially human, has not been well characterized. Thus, the aim of this study was to 1) identify EVs in human preimplantation embryos at different stages of their development using scanning and electron microscopy, and 2) investigate whether EVs can cross the zona pellucida (ZP) and be released from human embryos cultured in vitro. Human oocytes, zygotes, cleavage embryos and blastocysts donated for research were labeled with the tetraspanin EV marker CD9 and analyzed by scanning and transmission electron microscopy. Embryo culture conditioned media collected 3- and 5-days post fertilization were examined for the presence of EVs using electron microscopy. We detected numerous CD9 positive vesicles released from all embryos examined. They were observed on the surface of the plasma membrane, within the perivitelline space as well as throughout the zona pellucida. Interestingly, EVs were not seen in the ZP of all mature metaphase II oocytes, however, were detected just after fertilization in the ZP of zygotes and embryos. Electron microscopy using negative staining, and nanoparticle tracking analysis (NTA) of embryo conditioned culture media also showed the presence of vesicles of various sizes, which were round shaped, and had a lipid bilayer. Their size ranged from 30 to 500 nm, consistent with the sizes of exosomes and microvesicles. In conclusion, the results of the study provide evidence that human preimplantation embryos at all developmental stages secrete EVs into the perivitelline space, which then traverse through the ZP, and are then released into the surrounding culture medium. Abbreviations: EVs: extracellular vesicles; ZP: zona pellucida; CD9, CD63, and CD81: tetraspanin EV markers; NTA: nanoparticle tracking analysis; ESCRT: endosomal sorting complexes required for transport; SEM: scanning electron microscopy; TEM: transmission electron microscopy; TE: trophectoderm; ICM: inner cell mass; PVS: perivitelline space; MI: metaphase I; MII: metaphase II; GV: germinal vesicle; MVs/EXs: microvesicles/exosomes; hCG: human chorionic gonadotrophin; GnRH: gonadogrophin releasing hormone; ICSI: intracytoplasmic sperm injection; SPS: serum protein substitute; 1PN: one pronuclear zygote; 3PN: tri-pronuclear zygote; IgG: immunoglobulin G; PBS: phosphate buffer saline; ETHO: ethanol; ESED: Environmental Secondary Electron Detector; BSA: bovine serum albumin.

Serological and histological evaluation of species-specific immunocontraceptive vaccine antigens based on zona pellucida 3 in the small Indian mongoose (Herpestes auropunctatus).

The small Indian mongoose (Herpestes auropunctatus) was introduced to Japanese islands and has impacted on the island's biodiversity. Population control has been attempted through capturing but its efficiency has rapidly declined. Therefore, new additional control methods are required. Our focus has been on the immunocontraceptive vaccines, which act in an especially species-specific manner. The amino-acid sequence of the mongoose ovum zona pellucida protein 3 (ZP3) was decoded and two types of synthetic peptides (A and B) were produced. In this study, these peptides were administered to mongooses (each n=3) and the sera were collected to verify immunogenicity using ELISA and IHC. Treated mongoose sera showed an increasing of antibody titer according to immunizations and the antigen-antibody reactions against the endogenous mongoose ZP. In addition, IHC revealed that immune sera absorbed with each peptide showed a marked reduction in reactivity, which indicated the specificity of induced antibodies. These reactions were marked in peptide A treated mongoose sera, and the antibody titer of one of them lasted for at least 21 weeks. These results indicated that peptide A was a potential antigen, inducing autoantibody generation. Moreover, immunized rabbit antibodies recognized mongoose ZP species-specifically. However, the induction of robust immune memory was not observed. Also, the actual sterility effects of peptides remain unknown, it should be verified as a next step. In any case, this study verified synthetic peptides we developed are useful as the antigen candidates for immunocontraception of mongooses.

ZP2 pathogenic variants cause in vitro fertilization failure and female infertility.

PURPOSE: The oocyte-borne genetic causes leading to fertilization failure are largely unknown. We aimed to identify novel human pathogenic variants (PV) and genes causing fertilization failure. METHODS: We performed exome sequencing for a consanguineous family with a recessive inheritance pattern of female infertility characterized by oocytes with a thin zona pellucida (ZP) and fertilization failure in routine in vitro fertilization. Subsequent PV screening of ZP2 was performed in additional eight unrelated infertile women whose oocytes exhibited abnormal ZP and similar fertilization failure. Expression of ZP proteins was assessed in mutant oocytes by immunostaining, and functional studies of the wild-type and mutant proteins were carried out in CHO-K1 cells. RESULTS: Two homozygous s PV (c.1695-2A>G, and c.1691_1694dup (p.C566Wfs*5), respectively) of ZP2 were identified in the affected women from two unrelated consanguineous families. All oocytes carrying PV were surrounded by a thin ZP that was defective for sperm-binding. Immunostaining indicated a lack of ZP2 protein in the thin ZP. Studies in CHO cells showed that both PV resulted in a truncated ZP2 protein, which might be intracellularly sequestered and prematurely interacted with other ZP proteins. CONCLUSION: We identified loss-of-function PV of ZP2 causing a structurally abnormal and dysfunctional ZP, resulting in fertilization failure and female infertility.

Observations of follicle cell processes in a holocephalan.

The presence of follicular cellular processes (FCP) that cross the zona pellucida, has been recorded in the ovarian follicles of Callorhinchus callorhynchus. This constitutes the first report describing the presence of these structures in a species of the Holocephali. Considering that FCPs have only previously been reported in the Selachii, these findings suggest that FCPs could have been lost by the Batoidea after their divergence, around 280 M B.P.

The influence of ovarian hyperstimulation drugs on morphometry and morphology of human oocytes in ICSI program.

OBJECTIVE: To compare the influences of controlled ovarian hyperstimulation (COH) drugs using recombinant follicular stimulating hormone (rFSH) versus human menopausal gonadotropins (hMG) on morphometry and morphology of MII oocytes in ICSI cycles. MATERIALS AND METHODS: In this prospective study, 363 MII oocytes from 50 ICSI cycles with male factor infertility were evaluated. The patients were divided into two groups according to the protocols of COH: I- rFSH and II- hMG. The immature oocytes were excluded from the study. All oocytes were categorized into four morphological groups of normal, and those with single, double, or multiple defects. The inclusive morphometrical criteria were: areas and diameters of oocyte, ooplasm, and zona pellucida (ZP). Also, circumferences of oocyte and ooplasm were assessed. RESULTS: The ZP area and ooplasm diameter for both normal and abnormal oocytes were significantly higher in group I (P: .05; P: .028, respectively) compared to group II (P: .023; P: .003, respectively). In abnormal oocytes, ooplasm diameter was higher in group I compared to group II. Furthermore, ooplasm area for abnormal oocytes was significantly higher in group I compared to group II. There was an increasing trend for number of mature oocytes, in abnormal oocytes, for group I (5.53 ± 3.1) in comparison with group II (4.4 ± 2.97; P = .25). The rate of oocytes with normal morphology was significantly higher in hMG, when compared to rFSH groups. CONCLUSION: Morphometrical parameters were increased in rFSH group, but the normal morphology of oocytes were significantly enhanced in hMG group. Treatment with proper dosage of ovulation induction drugs may enhance the number of normal sized oocytes.

Is there any correlation between oocyte polarization microscopy findings with embryo time lapse monitoring in ICSI program?

PURPOSE: The aim was to investigate the relationship between the presence of the meiotic spindle (MS) and zona pellucida (ZP) birefringence of MII oocytes with morphokinetics variables of derived embryos in ICSI setting. METHODS: Using a polarization imaging system, the ZP birefringence and presence of MS were evaluated pre ICSI. Also, morphokinetics variables including time of second PB extrusion (tPB2), time of pronuclei appearance (tPNa), time of pronuclei fading (tPNf), time of two to eight discrete cells (t2-t8) ECC1 (t2-tPB2), cc2a (t3-t2), S2 (t4-t3) and S3 (t8-t5) as well as irregular cleavage events of 368 embryos were analyzed with time lapse monitoring (TLM). RESULTS: t5 occurred earlier in high birefringent ZP (HB-ZP) compared with low birefringent oocytes (LB-ZP; p = 0.001). In addition, t2 happened later in invisible MS compared to visible MS oocytes (p = 0.013). There were significantly lower rates of cell fusion (Fu) in oocytes with HB-ZP and also the Fu and trichotomous mitoses (TM) together in visible MS oocytes (p = 0.005, p = 0.001 and p = 0.001, respectively). CONCLUSIONS: Both t2 and t5 timings and irregular cleavage events of embryos were correlated with ZP birefringence and MS status, respectively. So, combining the information from both oocyte polarization microscopy imaging and embryo TLM can be a useful tool for single embryo transfer (SET) program.

Ultrastructural characteristics of human oocytes vitrified before and after in vitro maturation.

The development of an effective program that combines in vitro maturation (IVM) and cryopreservation for immature oocytes would represent a novel advance for in vitro fertilization (IVF), especially as a means to preserve the fertility of women in unique situations. The aim of this study was to analyze the ultrastructural characteristics of human oocytes, obtained after controlled ovarian stimulation, to determine whether IVM is best performed before or after vitrification. To this end, we analyzed the following features in a total of 22 MII oocytes: size, zona pellucida and perivitelline space, mitochondria number, M-SER (mitochondria-smooth endoplasmic reticulum) aggregates and M-V (mitochondria-vesicle) complexes, the number of cortical granules and microvilli, and the presence of vacuolization using transmission electron microscopy (TEM). Each oocyte presented a rounded shape, with an intact oolemma, and was surrounded by a continuous zona pellucida and perivitelline space. Statistical analysis comparing oocytes vitrified before or after IVM indicated that there were no significant differences between examined characteristics.

Automation and Optimization of Multipulse Laser Zona Drilling of Mouse Embryos During Embryo Biopsy.

Laser zona drilling (LZD) is a required step in many embryonic surgical procedures, for example, assisted hatching and preimplantation genetic diagnosis. LZD involves the ablation of the zona pellucida (ZP) using a laser while minimizing potentially harmful thermal effects on critical internal cell structures. OBJECTIVE: Develop a method for the automation and optimization of multipulse LZD, applied to cleavage-stage embryos. METHODS: A two-stage optimization is used. The first stage uses computer vision algorithms to identify embryonic structures and determines the optimal ablation zone farthest away from critical structures such as blastomeres. The second stage combines a genetic algorithm with a previously reported thermal analysis of LZD to optimize the combination of laser pulse locations and pulse durations. The goal is to minimize the peak temperature experienced by the blastomeres while creating the desired opening in the ZP. RESULTS: A proof of concept of the proposed LZD automation and optimization method is demonstrated through experiments on mouse embryos with positive results, as adequately sized openings are created. CONCLUSION: Automation of LZD is feasible and is a viable step toward the automation of embryo biopsy procedures. SIGNIFICANCE: LZD is a common but delicate procedure performed by human operators using subjective methods to gauge proper LZD procedure. Automation of LZD removes human error to increase the success rate of LZD. Although the proposed methods are developed for cleavage-stage embryos, the same methods may be applied to most types LZD procedures, embryos at different developmental stages, or nonembryonic cells.

The C-terminal region of OVGP1 remodels the zona pellucida and modifies fertility parameters.

OVGP1 is the major non-serum glycoprotein in the oviduct fluid at the time of fertilization and early embryo development. Its activity differs among species. Here, we show that the C-terminal region of recombinant OVGP1 regulates its binding to the extracellular zona pellucida and affects its activity during fertilization. While porcine OVGP1 penetrates two-thirds of the thickness of the zona pellucida, shorter OVGP1 glycoproteins, including rabbit OVGP1, are restricted to the outer one-third of the zona matrix. Deletion of the C-terminal region reduces the ability of the glycoprotein to penetrate through the zona pellucida and prevents OVGP1 endocytosis. This affects the structure of the zona matrix and increases its resistance to protease digestion. However, only full-length porcine OVGP1 is able to increase the efficiency rate of in vitro fertilization. Thus, our findings document that the presence or absence of conserved regions in the C-terminus of OVGP1 modify its association with the zona pellucida that affects matrix structure and renders the zona matrix permissive to sperm penetration and OVGP1 endocytosis into the egg.

Integrity of Narrow Epithelial Tubes in the C. elegans Excretory System Requires a Transient Luminal Matrix.

Most epithelial cells secrete a glycoprotein-rich apical extracellular matrix that can have diverse but still poorly understood roles in development and physiology. Zona Pellucida (ZP) domain glycoproteins are common constituents of these matrices, and their loss in humans is associated with a number of diseases. Understanding of the functions, organization and regulation of apical matrices has been hampered by difficulties in imaging them both in vivo and ex vivo. We identified the PAN-Apple, mucin and ZP domain glycoprotein LET-653 as an early and transient apical matrix component that shapes developing epithelia in C. elegans. LET-653 has modest effects on shaping of the vulva and epidermis, but is essential to prevent lumen fragmentation in the very narrow, unicellular excretory duct tube. We were able to image the transient LET-653 matrix by both live confocal imaging and transmission electron microscopy. Structure/function and fluorescence recovery after photobleaching studies revealed that LET-653 exists in two separate luminal matrix pools, a loose fibrillar matrix in the central core of the lumen, to which it binds dynamically via its PAN domains, and an apical-membrane-associated matrix, to which it binds stably via its ZP domain. The PAN domains are both necessary and sufficient to confer a cyclic pattern of duct lumen localization that precedes each molt, while the ZP domain is required for lumen integrity. Ectopic expression of full-length LET-653, but not the PAN domains alone, could expand lumen diameter in the developing gut tube, where LET-653 is not normally expressed. Together, these data support a model in which the PAN domains regulate the ability of the LET-653 ZP domain to interact with other factors at the apical membrane, and this ZP domain interaction promotes expansion and maintenance of lumen diameter. These data identify a transient apical matrix component present prior to cuticle secretion in C. elegans, demonstrate critical roles for this matrix component in supporting lumen integrity within narrow bore tubes such as those found in the mammalian microvasculature, and reveal functional importance of the evolutionarily conserved ZP domain in this tube protecting activity.

Site of Mammalian Sperm Acrosome Reaction.

Until recently, no special attention has been paid to the question of the site of mammalian sperm acrosome reaction (AR) in the female reproductive tract. Because AR is an essential process that enables the spermatozoon to fertilize, it is generally believed that it occurs at a specific step during sperm-egg interaction. It is generally thought that "the site of action coincides with the site of commitment." Thus, understanding the roles of AR and acrosomal substances is needed to gain insight into the site of the sperm commitment to undergo AR.

Association of high fetuin-B concentrations in serum with fertilization rate in IVF: a cross-sectional pilot study.

STUDY QUESTION: Is serum fetuin-B associated with the fertilization rate in in vitro fertilization (IVF)? SUMMARY ANSWER: Serum fetuin-B increased during IVF cycles when oocytes could be fertilized while remained unchanged in fertilization failure. WHAT IS KNOWN ALREADY: Fetuin-B deficiency in mice causes premature zona pellucida hardening mediated by the zona protease ovastacin. Thus fetuin-B deficiency renders females infertile. STUDY DESIGN, SIZE, DURATION: We determined the human serum fetuin-B reference range, studying longitudinally, over the course of one month, five male and seven female volunteers without hormone treatment and four female volunteers on varying hormonal contraception. We sampled blood and determined serum fetuin-B, luteinizing hormone (LH), estradiol (E2) and progesterone (P4). In addition, we determined serum fetuin-B and estradiol in eight women undergoing intracytoplasmatic sperm injection (ICSI, nine ICSI cycles) and 19 women undergoing IVF (21 IVF cycles) after ovarian stimulation with recombinant human follicular stimulating hormone (rFSH) and/or a combined medication of FSH and LH. At least three blood samples were analyzed in each cycle. We compared serum fetuin-B and follicular fluid fetuin-B in nine patients by measuring follicular fetuin-B in pooled follicular fluid, and in fluid obtained from individual follicles. Samples were drawn from January 2012 to March 2014. PARTICIPANTS/MATERIALS, SETTING, METHOD: All volunteers and patients gave informed consent. Fetuin-B was measured employing a commercial sandwich enzyme-linked immunosorbent assay. Serum fetuin-B was determined as duplicates in 5 male (34 ± 14.6 years) and 11 female volunteers (29.4 ± 4.1 years) as well as in female volunteers on hormonal contraception (30.0 ± 6.5 years). The duplicate standard deviation was 4.0 ± 2.3%. The contraceptive drugs were mono or combined preparations containing 0-0.03 mg ethinyl estradiol, and 0.15-3.0 mg of various progestins. In addition, serum fetuin-B was determined as triplicates in 27 female patients undergoing conventional IVF (19) or ICSI (8). The triplicate standard deviation was 3.3 ± 1.8%. IVF was declared as 'successful', if at least one oocyte was fertilized, and 'unsuccessful', if no oocyte could be fertilized. Patient age was 34.4 ± 4.4 years in successful IVF, and 35.4 ± 3.3 years in unsuccessful IVF. Serum and follicular fluid of patients undergoing controlled ovarian hyperstimulation were analyzed. Serum was drawn at the day of follicle aspiration. MAIN RESULTS AND THE ROLE OF CHANCE: Serum fetuin-B and follicular fluid fetuin-B were not significantly different in six out of nine patients suggesting, in principle, free exchange of fetuin-B between serum and follicular fluid. Thus serum fetuin-B may be used as a proxy of follicular fluid fetuin-B. Serum fetuin-B increased during successful IVF cycles (n = 15, P < 0.0001), but did not change in unsuccessful IVF cycles (n = 6, P = 0.118) despite increased estradiol levels (P = 0.0019 and P = 0.0254, respectively). LIMITATIONS, REASONS FOR CAUTION: The female volunteers self-reported their respective hormone medication. Medication was verified by serum estradiol, LH and progesterone measurements. For oocyte harvesting, the vaginal wall was punctured once only to minimize co-morbidity. Low grade cross-contamination of individual follicular fluid aspirates and contamination of the follicular fluid with small amounts of blood were inevitable. Samples were routinely checked for the presence of hemoglobin that would suggest blood contamination. Only samples containing <250 erythrocyte equivalents/µl were used for analysis. WIDER IMPLICATIONS OF THE FINDING: Serum fetuin-B may be used as a marker to predict the fertilization success in IVF. Fetuin-B levels attained during IVF stimulation may help to make an informed decision whether oocytes should be fertilized by IVF or by ICSI to overcome the zona pellucida as a barrier. STUDY FUNDING/COMPETING INTERESTS: The research was supported by a grant from Deutsche Forschungsgemeinschaft and by the START program of the Medical Faculty of RWTH Aachen University. J.F., E.D., J.N., B.R. and W.J.-D. declare that they are named inventors on the RWTH Aachen University patent application EP 13157317.2, 'Use of fetuin-B for culture of oocytes', applied for by RWTH Aachen University.