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Selection for less efficient egg transfer in Human Botfly life cycle

Selection for less efficient egg transfer in Human Botfly life cycle


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I have heard that the Human Botfly transfers its eggs through other invertebrates, and it strikes my curiosity that if an insect could simply land on its host directly to deposit its eggs, then why have intermediate hosts?

I have speculated that the vectors may be better specialized for penetrating hosts, and that penetrating hosts may be difficult, or that approaching hosts is risky. Another speculation on my part is that the Human Botfly may be filling a niche where it does not need to compete.


In multi-host complex life cycles (CLCs), an intermediate host often aids in the dispersal of offspring.

In general terms, an animal, say a botfly, have an ultimate "purpose" or 'goal' to spread their genetic information as much as possible. This could mean producing a large number of offspring and hoping some survive or it could mean having one offspring, investing a lot of energy and ensuring it does survive. Regardless, spreading genetic information often requires offspring to disperse (or be spread) a great distance, which may allow the individuals' genetic information to dominate an new region.

In the case of botflies, having an intermediate host means the genetic information can be spread a great distance.

If you're looking for a more thorough explanation of complex life cycles, this paper describes it well. There is also this site more specific to the botfly which is not peer reviewed, but well-researched and descriptive.


it makes sense because the mosquito which is usually the intermediate host is more "sneaky" than the bot fly. so its better for the survival of the bot fly and better for the transfer of the egg. The egg hatches upon contact with the hosts skin so its not inserted by the mosquito bite. the hatched larva diggs itself into the skin then.


Elective single embryo transfer- the power of one

Despite the highest historical live birth success rates for couples undergoing in vitro fertilization (IVF), there has been an epidemic of iatrogenic twin and higher order gestation conceived from this treatment. Continued improvement in cryopreservation techniques have allowed preservation of supernumerary embryos for use in future cycles, and refinements in culture systems and embryo selection have resulted in the transfer of fewer embryos while maintaining favorable pregnancy rates. The voluntary transfer of a single high quality embryo, elective single embryo transfer (eSET), has significantly reduced multiple gestation rates and maximized the rate of singleton pregnancy without compromising overall success rates. Although eSET is the standard of care in several developed countries, utilization in the United States has been slow. States with mandated IVF insurance have seen decreases in preterm birth rates yielding down stream health care savings. Herein, the evolution and future applications of this practice to reduce the risk of iatrogenic twins is reviewed.


Introduction

Bryophytes – comprising mosses, liverworts and hornworts – represent three out of the four ancient lineages of land plants (Fig. 1). These early diverging land plants are distinct from other land plant lineages, as they are non-vascular. Furthermore, they use free-swimming motile sperm for fertilization, an attribute shared by the fern lineage but lost in seed plants. In addition, moss plants have a relatively simple morphology, with many fewer cell fates than in flowering plants. Interestingly, gene families encoding much of the basic developmental `tool kit' identified in flowering plants are conserved in the genome of the moss Physcomitrella patens (Floyd and Bowman, 2007 Rensing et al., 2008). During the nearly half billion years since diverging from the moss lineage, flowering plants appeared to have re-purposed much of this tool kit as new developmental processes have evolved.

Using the powerful tools afforded by efficient gene targeting in P. patens (Kammerer and Cove, 1996 Schaefer and Zryd, 1997 Strepp et al., 1998), it has become possible to study the roles of many of these genetic pathways in moss. In doing so, we can begin to decipher which developmental mechanisms are elaborations of basic mechanisms that were present in ancestral land plants and which represent novel innovations in the flowering plant lineage. Several P. patens developmental pathways will be particularly interesting to dissect in light of what might be learned about important events in land plant evolution.

In this primer, we introduce the P. patens life cycle and explain how aspects of this life cycle make this particular moss especially suited for molecular genetic experiments, including targeted deletions, allele replacements and RNA interference (RNAi). In addition, we summarize key findings in P. patens in the past 5 years that have propelled the plant developmental field forward.


Melatonin and Selection of a Sexual Mate

Some notable new findings suggest that melatonin may have a role in the selection of a sexual partner. Bertrand and colleagues [ 48] observed that administering melatonin (50 μg/ml) to male zebra finches (Taeniopygia guttata) in the drinking fluid in combination with carotenoids (100 μg/ml) enhanced the brightness of the carotenoid-based pigmentation in their bills. Given that males with brighter colored bills (assessed using a Dulux Trade [Slough, Berkshire, England] color chart) are more likely to be selected as a mate by a female zebra finch, melatonin may aid in the selection of a mate. Like melatonin [ 49, 50], carotenoids are free radical scavengers and antioxidants [ 51, 52]. To protect against oxidative stress, nonpigmentary melatonin in zebra finches is possibly used preferentially in the detoxification of radicals over the carotenoids, leaving the latter available to deposit in the bill and thereby improving its color and enhancing the chance of that male being selected as a sexual partner.

In the study by Bertrand et al. [ 48], the amount of drinking fluid consumed by the finches was not measured, so the daily intake of melatonin could not be calculated. Although it seems likely that the quantity of melatonin consumed would have caused pharmacological levels in the blood of the birds, this remains unknown without information on the amount normally absorbed from the gastrointestinal tract of the zebra finch. Had plasma levels of melatonin been measured, an answer to the issue of pharmacological and physiological levels could likely have been obtained. There are wide variations in melatonin concentrations in different fluids [ 53, 54], so what is a pharmacological level in one fluid is a physiological level in another.

Given its actions beyond those as a reducer of oxidative stress, it is also possible that melatonin may influence bill color by means that do not directly involve preservation of the carotenoids (e.g., its effects on metabolism or the immune system, as discussed elsewhere [ 48]). Nevertheless, if pigmentation of the bill underlies sexual attractiveness [ 55], the final effect of melatonin would be the same.

Melatonin could theoretically assist in determining the selection of a sexual partner by finches in another way. Melatonin receptors were identified in neurons of two central sensorimotor areas (nucleus hyperstriatalis pars caudale and nucleus robustus archistriatalis) of the descending song control circuit that are essential for the song pattern of male zebra finches [ 56]. Melatonin receptors were identified in the peripheral hypoglossal nuclei, which are likewise part of the song circuit. When melatonin was applied to brain slices containing neurons of the nucleus robustus archistriatalis of the song control circuit, the firing rate of the neurons was suppressed, suggesting that melatonin may alter the singing ability of the bird. When a melatonin receptor antagonist (S 20928) was systemically administered in vivo to male finches at the beginning of the night just before the nocturnal melatonin rise, it shortened both the song and the motif length and influenced the song syllable lengths the following day. However, the temporal pattern of singing was not altered, indicating that the disrupted song produced by the melatonin receptor antagonist was not merely a circadian disturbance. Given that female finches are attracted by long song and motif lengths, these data are the first to imply that melatonin may have a role in male courtship behavior. The findings are notable because the ability of the male to sing makes it a preferred partner for sexual activity [ 57]. In this study, it was difficult to test the effect of melatonin directly in vivo because endogenous melatonin is available nightly. To circumvent this, the investigators would have had to have pinealectomized the birds. In lieu of this, they administered a membrane melatonin receptor blocker.

In fish, physiological color changes for the purpose of attracting mates is commonplace. Sköld and coworkers [ 58] investigated the mechanisms governing nuptial coloration in the two-spotted goby (Gobiusculus flavescens). Female gobies develop an orange belly that acts as an ornament to attract members of the opposite sex the color is a consequence of the pigmentation of the gonads in combination with chromatophore-rich pigmentation and transparency of the skin. The pigmentation can change rapidly, and when the bright color is on the background of the darker surrounding skin, the belly appears to glow. When skin explants of gobies were treated with a combination of either melatonin and melanocyte-stimulating hormone or melatonin and prolactin, the orange coloration and transparency of the belly skin were exaggerated. The authors noted that this duplicates the chromatic glow that accompanies courtship. The nuptial coloration change induced by melatonin and other hormones would presumably benefit the individual in terms of attracting a sexual mate. While the findings by Sköld et al. are consistent with the hypothesis that melatonin and other hormones are relevant to sexual selection, their investigations were performed on belly skin explants whether this translates into the same function for those hormones in vivo remains to be tested.

The zebra finch and the goby are only two of the numerous species that use ornamental pigmentation to attract a mate. Colorful plumage generally signals superior genetic quality and is a common ploy used by many bird species as a sexual attractant [ 59], while bright pigmentation of certain body areas serves this purpose in some reptiles, amphibians, and mammals. On the basis of the studies already summarized, it may be worthwhile to examine more broadly the role of melatonin in the extravagant pigmentary changes that serve as a sexual enticement and represent genetic quality in a variety of species. There is ample precedent for melatonin's working at the level of the integument. Melatonin receptors exist in mammalian skin and hair follicles [ 60, 61], and the indole modulates hair growth, pigmentary changes in fur, and molting in many mammalian species [ 60], as well as scale growth in fish [ 62]. Because crypsis and mimicry both require clever skin or hair pigmentary adaptations, melatonin could be examined relative to these physiological changes as well.


Abstract

This review offers an overview of the basic characteristics of in vivo embryo technologies, their current status, the main findings and the advances gained in recent years, and the outstanding subjects for increasing their efficiency. The use of superovulation and embryo transfer procedures remains affected by a high variability in the ovulatory response to hormonal treatment and by a low and variable number of transferable embryos and offspring obtained. This variability has been classically identified with both extrinsic (source, purity of gonadotrophins and protocol of administration) and intrinsic factors (breed, age, nutrition and reproductive status), which are reviewed in this paper. However, emerging data indicate that the main causes of variability are related to endocrine and ovarian factors, and so the number of studies and procedures addressing a better understanding and control of these factors may be increased in the future. The accomplishment of this objective, the improvement of procedures for embryo conservation and for the selection and management of recipient females, will allow further development and application of this technology.

Extra keywords: embryo cryopreservation, follicle dynamic, luteal function, superovulation.


Presence on Human Microbiome

D. folliculorum localize on the human face, feeding on skin cells, sebum, and hormones. They inhabit hair follicles, particularly those of the eyelashes and eyebrows. Several mites can inhabit the same follicle and will climb to the opening in order to mate or transfer hosts. Infants typically acquire D. folliculorum from physical contact with their mothers, however, the lack of sebum production in young children and adolescents prevents D. folliculorum from colonizing efficiently. As humans reach adulthood, sebum production increases substantially, peaking at ages 20-30. Therefore, although D. folliculorum are rarely found on children less than 5 years old, they are present on every adult that has been tested. D. folliculorum have been shown to colonize more on men than on women. Additionally, infestation tends to be the worst in the elderly and those with immunodeficiency. ⎙]

Conflicting sources characterize D. folliculorum as either commensals or parasites of their human hosts. In the majority of cases, the presence of D. folliculorum has no harmful effects. However, in some cases infestation can lead to serious medical conditions such as acne rosacea and marginal blepharitis. ⎚] Demidicosis or demodicidosis are blanket terms for diseases caused by Demodex mites. Demodicosis rosacea includes symptoms of, “Dryness, follicular scaling, superficial vesicles, and pustules.” ⎛] The cause is tied to behaviors of the human host that encourage the mites’ replication, such as neglecting to wash one’s face or using creamy or oily products. These behaviors lead to a build up of lipids, which D. folliculorum and D. brevis feed off of. ⎜] In Blepharitis, bacteria carried by Demodex triggers severe immune responses in the host. Symptoms include, “Cylindrical dandruff, disorders of eyelashes, lid margin inflammation,” in addition to, “Itching, burning, foreign body sensation, crusting and redness of the lid margin, and blurry vision.” ⎝] The inflammation induced by the bacteria may manifest in the conjuctiva or the cornea of the eyes, leading to further damage. ⎞] Immunodeficiency has been shown to be a factor in the the infestation of D. folliculorum and D. brevis. ⎟]


The Embryo Project Encyclopedia

Green fluorescent protein (GFP) is a protein in the jellyfish Aequorea Victoria that exhibits green fluorescence when exposed to light. The protein has 238 amino acids, three of them (Numbers 65 to 67) form a structure that emits visible green fluorescent light. In the jellyfish, GFP interacts with another protein, called aequorin, which emits blue light when added with calcium. Biologists use GFP to study cells in embryos and fetuses during developmental processes.

Biologists use GFP as a marker protein. GFP can attach to and mark another protein with fluorescence, enabling scientists to see the presence of the particular protein in an organic structure. Gfp refers to the gene that produces green fluorescent protein. Using DNA recombinant technology, scientists combine the Gfp gene to a another gene that produces a protein that they want to study, and then they insert the complex into a cell. If the cell produces the green fluorescence, scientists infer that the cell expresses the target gene as well. Moreover, scientists use GFP to label specific organelles, cells, tissues. As the Gfp gene is heritable, the descendants of labeled entities also exhibit green fluorescence.

Edmund N. Harvey, a professor at Princeton University in Princeton, New Jersey, initiated the studies on bioluminescence in the US. In 1921, Harvey described the yellow tissues in the umbrella of jellyfish as being luminous in particular conditions, such as at night or when the jellyfish is stimulated with electricity. In 1955, Demorest Davenport at the University of California at Santa Barbara in Santa Barbara, California, and Joseph Nicol at Plymouth Marine Laboratory in Plymouth, England, used photoelectric recording and histological methods to confirm Harvey's descriptions, and they identified the green fluorescent materials in the marginal canal of the umbrella.

In the same year, Osamu Shimomura became a research assistant at Nagoya University in Nagoya, Japan, and he crystallized the luciferin, a light-emitting compound found in the sea-firefly Vargula hilgendorfii. Shimomura published his results in 1957. One of Harvey's students, Frank H. Johnson, studied bioluminescence at Princeton University. Johnson followed Shimomura's work and invited him to work in the US, and in 1960 Shimomura received a Fulbright Travel Grant and started working with Johnson. Shortly after Shimomura arrived in the US, Johnson introduced the bioluminescence of Aequorea Victoria to Shimomura. In the US, jellyfish live only on the west coast, so Shimomura traveled to the Friday Harbor Laboratories of the University of Washington in San Juan Island, Washington, during the summer of 1961. After catching about 10,000 jellyfish, Shimomura took the extracts of the jellyfish and preserved it in dry-ice to bring it back to Princeton in September of 1961.

At Princeton, Shimomura and his colleagues started to purify the bioluminescent substance, and they found that it was a protein, which they called aequorin. When they purified aequorin, they also discovered traces of another protein, which showed green fluorescence. Shimomura's team published the findings in "Exraction, Purification, and Properties of Aequorin" in 1962. The paper was about aequorin, but it also described a green protein, which exhibited green fluorescence under sunlight. John W. Hasting and James G. Morin, who later researched aequorin, termed the protein as green fluorescent protein in 1971.

Shimomura focused on aequorin, purified the protein, crystallized it, and elucidated its underlying structure. He also studied the properties of GFP, and published his last paper on GFP in 1979. In 1981, after leaving Princeton University for the Marine Biology Laboratory in Woods Hole, Massachusetts, Shimomura did not research on GFP anymore. From 1979 to 1992, many researchers studied various aspects of GFP, including the use of Nuclear Magnetic Resonance to study the amino acids of the protein, the use of X-rays to study its crystal, and the evolution of GFP.

In the early 1990s, molecular biologist Douglas Prasher, at the Marine Biology Laboratory, used GFP to design probes, a technology involving fragments of DNA to detect the presence of nucleotide sequences. Prasher isolated the complementary DNA (cDNA) of Gfp gene, and he published the sequence of the gene in 1992. After the publication of the cDNA sequence in 1992, Prasher's funding from the American Cancer Society in Atlanta, Georgia, expired. When he applied for funding from the US National Institute of Health in Bethesda, Maryland, the reviewer argued that Prasher's research lacked contributions to society. As Prasher could not secure funding to support his research any further, he left the Marine Biology Laboratory to work for the US Department of Agriculture in Massachusetts.

After Prasher's publication in 1992, many scientists tried to transfer and express the Gfp gene in organisms other than jellyfish using DNA recombinant technology, and Martin Chalfie was the first who succeeded. Chalfie, a Professor at Columbia University in New York, New York, studied the development of the nematode Caenorhabditis elegans. Chalfie heard about the protein GFP in a lecture, and he speculated that GFP might facilitate his study of gene expression in C. elegans. Chalfie's team obtained the cDNA of the gene Gfp from Prasher and inserted only the coding sequence of Gfp gene first in the bacterium Escherichia Coli, and then in C. elegans. Chalfie and his team found that Gfp gene produced GFP without added enzymes or substrates in both organisms. In 1994, Chalfie published his results in "Green Fluorescent Protein as a Marker for Gene Expression". The detection of GFP needed only ultraviolet light. Thereafter, many biologists introduced GFP into their experiments to study gene expression. Satoshi Inouye and Frederick Tsuji at Princeton University also expressed Gfp in E. Coli in 1994.

Many scientists tried to mutate the Gfp gene to make the resultant protein react to wider wavelengths and emanate different colors. Other scientists studied different fluorescent proteins (FPs). Roger Tsien, a professor at the University of California San Diego, in San Diego, California, reengineered the gene Gfp to produce the protein in different structures. His team also reengineered other FPs. Due to Tsien's and other bioengineers' efforts, GFP could not only exhibit brighter fluorescence, but also respond to a wider range of wavelengths, as well as emit almost all colors, except for red. Tsien's findings enabled scientists to tag multiple colored GFPs to different proteins, cells, or organelles of interest, and scientists could study the interaction of those particles. Red FP became available in 1999, when Sergey Lukyanov's team at the Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry in Moscow, Russia, found that some corals contained the red fluorescent protein, called DsRed. Other laboratories developed fluorescent sensors for calcium, protease and other biological molecules. Since then, scientists have reported more than 150 distinct GFP-like proteins in many species.

As GFP does not interfere with biological processes when used in vivo, biologists use it to study how organisms develop. For example, after 1994, Chalfie and his colleagues applied GFP in the study of the neuron development of C. elegans. In a 2002 paper, Chalfie and his colleagues describe how they first labeled a specific gene involved in tactile perception in neuron cells with GFP, and then observed the amount of fluorescence emitted by those cells. Because mutant cells produced less or more GFP than normal cells, the abnormal amount of fluorescence production indicated the abnormal development of mutants. Since then, this field of research expanded to many other organisms, including fruitflies, mice, and zebra fish.

On 10 December 2008, The Royal Swedish Academy of Science academy awarded the Noble Prize in Chemistry to Tsien, Chalfie, and Shimomura for their discoveries on GFP.


Selection for less efficient egg transfer in Human Botfly life cycle - Biology

Reproductive success is measured primarily by pigs produced per sow per year and is dependent upon both farrowing rate and litter size. In order to achieve optimal reproductive rates, both the anatomical and physiological workings of the reproductive system must function properly. A basic understanding of the anatomical and physiological function of the female pig reproductive system can aid producers in anticipating and troubleshooting reproductive problems, and in facilitating decisions which impact performance of the breeding herd. This article introduces the reader to the anatomy and physiology of female reproduction and how this acts to enhance or inhibit performance.

The Female Reproductive Tract

General Parts and Support

The female reproductive tract is composed of paired right and left ovaries, oviducts, and uterine horns (Figure 1). It contains only a single cervix, vagina and vulva (external genitalia). Collectively, these structures are supported by the broad ligament and hang loosely suspended below the rectum in the both pelvic canal and lower abdomen. The broad ligament is made of tough connective tissue, attaching near the point of the spine, and running continuously with the inner most layer of the abdominal cavity. Many of the blood vessels and nerves travel through this large piece tissue in order to supply the reproductive tract with blood, hormones and neural stimuli. In prepubertal gilts, the ligament is short, paper thin, and almost transparent. However, in late pregnancy it becomes very long as it stretches and thickens in order to support the increasing weight of the pregnant reproductive tract.

The Ovary

Follicle Development

The ovary of the pig is primarily important because it is the source for both reproductive hormones and eggs. The ovary is particularly responsive to important hormones that are released from other organs, especially those of the pituitary. The pituitary is located near the base of the brain and is the source of Follicle Stimulating Hormone (FSH) and Luteinizing Hormone (LH). It is these two hormones which are responsible for initiating and stimulating the ovary to become active in order to begin reproduction. Note: PG600Ò is an approved drug for stimulating estrus in gilts and is very close in structure and function to FSH and LH. FSH causes many small follicles (< 3 mm in diameter) to grow into medium sized follicles (3 - 6.5 mm). These follicles appear as small, blister-like structures on the surface of the ovary (Figure 1). Each follicle contains an egg and produces considerable amounts of steroid hormones, most notably, estrogen. The other pituitary hormone, LH, is important for the continued growth of the medium sized follicles into large follicles, which are responsible for releasing the egg at estrus. As the follicles grow, the egg inside the follicle also begins to mature as estrogen levels inside the follicle become very high. This elevated follicle estrogen ultimately leads to increased estrogen levels in the blood. When the blood concentrations of estrogen become high enough, the female shows signs of estrus and eventually stands for the back pressure test in the presence of a boar.

Ovulation

The occurrence of peak levels of estrogen in the blood, which originate from the large follicles on the ovary, is followed closely by a surge of LH into the blood at the time of estrus. Ovulation of the large follicles appears to occur at a specific time interval (

42 h) after this LH surge. The eggs from all of the large follicles from both ovaries ovulate in a relatively short period of time (

3 h). However, even though there is minimal variation in the time to ovulate all follicles within a female, the time of ovulation after onset of estrus is highly variable between females. Some sows are observed to ovulate as early as 24 h to as late as 60 h after onset of estrus. This variation in time of ovulation is greater than or equal to 24 h. Therefore this variation is very significant since after ovulation, the egg only lives 8-12 h, and sperm are less able to fertilize an egg 24 h after insemination. The objective then, for obtaining optimal reproductive performance, is to inseminate females within 12 h before ovulation. It has been observed that females ovulate approximately two-thirds of the way through the length of estrus. However, since length of estrus, and time of ovulation after onset of estrus are variable, and knowing when the female is out of estrus is not of any practical value, the best way to compensate for variation in time of ovulation is to get the female bred for the potential of both and early and late ovulation, by using two inseminations 12 to 24 h apart. A note of caution: these numbers are only estimates, and sperm life may be even shorter as stored extended semen ages.

Ovulation Rate

Ovulation rate or the number of large follicles that grow and ovulate (release eggs) at estrus is important because this number becomes the first limiting factor to litter size in pigs. Interestingly, the pig develops hundreds of small follicles during an estrous cycle, but typically only ovulates between 10-20 of these follicles at estrus. Some abnormalities do occur with follicle development, which ultimately leads to abnormal ovulation rates. In gilts at pubertal estrus, ovulation sometimes does not occur or only a few follicles actually grow and ovulate. This is also observed in some sows after weaning at certain times of the year and under cases of excessive weight loss during lactation. Sometimes follicles develop to the large size but fail to ovulate, leading to the condition known as cystic ovary disease. There have been reports where certain factors have been shown to specifically increase ovulation rate. In gilts, elevated feeding of dietary energy during the last 10 days before estrus and an increase in the number of lifetime estrous cycles appears to increase ovulation rate in females.

Post-Ovulation

Once the follicle ovulates and releases the egg, there is some bleeding from the rupture site but this quickly forms a small blood clot on the ovary where the follicle once was. Within a few hours after ovulation, the cells of the follicle begin to rapidly change and divide into a new type of cell, which over the next few days, will form into corpus luteum cells, which produce progesterone, the hormone needed for developing the uterus, inhibiting estrus, and maintaining pregnancy. After ovulation of all the large follicles at estrus, the egg is moved into the tube called the oviduct. This movement occurs by the coordinated muscular contractions of a thin piece of tissue called the fimbria (Figure 1). The fimbria wraps around the entire ovary, and under the influence of estrogen, induces muscular contractions which propel all of the eggs into the funnel shaped opening of the oviduct.

The Oviduct

The oviduct is a short convoluted tube that connects the ovary to the uterus (Figure 1). This short tube is very important since it must propel the eggs in one direction and the sperm in the opposite direction. Fertilization takes place in the mid portion of the tube, called the ampulla. Most of the eggs reach the site of fertilization within 30 min to 1 h after ovulation. The eggs will remain viable and fertilizable in the tube for approximately 8-12 h after ovulation. Therefore it is important that insemination occur prior to ovulation so that sperm are waiting for the egg.

Insemination and Fertilization

Sperm are typically deposited into the reproductive tract near the junction of the cervix and uterus. Although much of the inseminate enters the uterus, there is a significant back-flow loss of semen volume which occurs over the next several hours after insemination. Under stimulation from uterine contractions induced by both oxytocin from the female and prostaglandins in the semen of the boar, sperm are moved to the site of fertilization. Of the billions of sperm that are inseminated, only a very small fraction of these (hundreds to thousands) actually arrive at the site of fertilization. Many sperm are prevented from entering the oviduct from the uterus by a small restrictive muscle that can open and close under the control of hormones. Therefore, only a small sperm reservoir is typically maintained in the oviduct. Some sperm cells reach the site of fertilization within minutes after insemination, however, these sperm cells are incapable of fertilizing eggs because a time dependent passage through the female uterus is required to prepare them to attach to the egg. Most of the fertilizing sperm will reach the egg in about 3-6 h after insemination, but numbers of sperm will continue to increase in the oviduct for up to about 12 h after insemination.

It is important to note that the levels of estrogen and progesterone can affect the movement of the sperm and eggs in the oviduct. Excessive estrogen at this time has been reported to cause egg retention in the oviduct, while excessive progesterone has the effect of opening the oviduct and speeding entry of the sperm and exit of the eggs. Once fertilization does occur, early embryos develop in the oviduct for

48 h until they leave the oviduct. Eggs that are not fertilized usually pass into the uterus within several days. Sperm that are in the oviduct and uterus which do not fertilize eggs are destroyed by cells of the immune system over the next few days.

The Uterus

The uterus is the largest single portion of the female reproductive tract and is capable of considerable change in size from the non-pregnant to the pregnant state. The uterus is composed of paired uterine horns with the cervix at one end and the oviducts at the other. The uterus has four layers, an inner most layer which is glandular, two additional inner layers which are muscular, and the fourth layer, which forms the outer surface. The large muscle layers are important for propelling sperm to the oviduct, moving and spacing embryos before attachment, and for delivery of piglets at farrowing. These layers are responsive to many of the reproductive hormones and how and when contractions will occur depend upon which hormones are present. The other important layer is the inner most glandular layer which produces and secretes hormones and nutrients for the developing embryos.

Pregnancy

After fertilization, the early embryos enter the uterus on day 4 and remain free-floating and mix with each other until approximately d 12-13. Fertilization of all eggs is usually very high (95%) and therefore many embryos actually enter the uterus. However, not all embryos are equal and some are defective and others are slower developing. It has been observed that many embryos are lost prior to day 10 of pregnancy and additional embryos are lost prior to day 20, for a total embryo loss of almost 40% prior today 20 of pregnancy. Obviously, this is one of the primary factors limiting litter size in swine. Of the embryos that do survive past day 10 of pregnancy, they must signal the mother that they are present by secreting the hormone estrogen, which in turn will prevent the mother from releasing prostaglandin from the uterus, a hormone which will destroy the corpus luteum and cease progesterone production, resulting in termination of pregnancy. If there are no embryos present or too few embryos produce a signal, then prostaglandin is released and this has the effect of reducing blood supply to the corpus luteum. Once progesterone is suppressed, uterine contractions may begin and a new wave of follicles begins to develop, so that the female can begin to cycle again. If the embryos are successful at signaling the mother, progesterone will remain high and uterine contractions will be inhibited until birth. If females do not conceive, they should return to estrus at a regular interval (21 days). However, if their eggs are fertilized but pregnancy cannot be established, then the female will often return to estrus at irregular intervals after mating. If pregnancy is established, the embryos will begin attachment to the lining of the uterus between d 14-17. They will loosely attach throughout the uterine surface and will continue to strengthen their attachment to allow more efficient transfer of nutrients. The uterus will accommodate many more embryos and fetuses than can actually be supported to term, until d 30. However, after this day, the fetuses that cannot be supported, due to a limit in uterine size, will be lost before day 50 of pregnancy.

The Cervix

The cervix is approximately one inch in diameter and about 6-8 inches in length, and connects the vagina and the uterus. It is made of tough connective tissue and contains limited amounts of glandular and muscular tissue. It contains a series of five interdigitating pads (Figure 1) which provide pressure points for locking of the penis (or AI catheters). Its primary functions are to serve as a locking mechanism for the penis. The cervix is also a flexible structure and can open and close under the influence of hormones. The cervix is important for protecting the fetuses and will remain tightly closed except at estrus and at farrowing, when it will dilate to accommodate the boar's penis and to allow passage of the piglets through the birth canal. The cervix is also the primary source of mucus. Under estrogen stimulation, such as that which occurs at estrus, the mucus becomes watery and can sometimes be seen seeping from the vulva. This mucus serves as a lubricant for the penis of the boar. Under progesterone stimulation during pregnancy, the cervical mucus will thicken and form a plug to prevent any contaminants from entering the sterile uterine environment. This cervical plug will dissolve just prior to farrowing.

The Vagina

The vagina is approximately 12-18 inches long and connects the cervix to the external genitalia of the pig. There is limited muscular and glandular tissue in the vagina and it serves primarily as a copulatory organ for the boar and as a passageway from the uterus to the outside. The vagina does have some immunoprotectant function since antibodies such as IgA are present to prevent any uterine contamination. The pH of the vagina is also acidic and is unfavorable to sperm and microbe survival.

The bladder empties into the vagina on the floor of the vagina approximately two inches from the external opening. This is important because many types of spiral AI catheters can mistakenly be inserted into the urethral opening of the bladder. The vagina also houses the clitoris, an organ analogous to the male penis, which when stimulated may induce hormones responsible for initiating muscular contractions in the reproductive tract.

The Vulva

The external genitalia of the female pig is composed of some connective and fatty tissues. The vulva is endowed with blood vessels and in gilts the vulva is often observed to swell and change color near the time of estrus. This swelling and color change are not as evident in sows, and color changes are not observable in dark skinned pigs.


Social egg freezing: A viable option for fertility preservation

ABSTRACT: Social egg freezing is now a viable option for women in our society who wish to preserve their fertility and delay childbearing. Over the past few years, substantial advancements have been made in the egg freezing technique. Evidence from randomized controlled trials has demonstrated that frozen eggs can work as well as fresh ones for in vitro fertilization. As a result, egg freezing is no longer considered experimental and the procedure has grown in popularity and gained mainstream attention. The use of this technology is driven by the fact that women are choosing to have children much later in life for a variety of reasons, the most common in one study being the lack of a partner. It is important for health care providers to educate women about the risks that come with delayed childbearing: infertility, aneuploidy, and miscarriage. Despite these and other concerns, including the false sense of security that social egg freezing can create, this technology is the best option when attempting to provide women with reproductive longevity more like that enjoyed by men.

While cryopreservation of eggs is no longer considered experimental and is being offered as an employment benefit by some large organizations, it is important for women to understand that no number of stored eggs will guarantee a baby in the future.

Egg freezing has come a long way since the first birth from a cryopreserved human oocyte was reported in the Lancet in 1986.[1] Initial attempts utilized a technique called slow-freezing that yielded eggs with poor survival and pregnancy rates of approximately 12% per embryo transferred.[2] As a result, egg freezing was generally reserved for women in countries where embryo storage was not permitted by law or in cases where a single woman undergoing potentially sterilizing treatments wished to preserve her fertility.[3,4] An example of this would be a woman who banked her eggs before being treated for breast cancer with cyclophosphamide, a chemotherapeutic agent toxic to the ovaries.[3] So even though the older techniques meant that 100 eggs might be needed to achieve one live birth, low success rates were acceptable given the bleak alternative of sterility for cancer patients.[3,5] More recently, following substantial advancements in cryopreservation technique and evidence from randomized controlled trials, egg freezing has received mainstream attention.[6-9] Pregnancy rates with frozen eggs now match those achieved with fresh eggs, and elective fertility preservation is an exciting new frontier in reproductive medicine.

Social egg freezing refers to the cryopreservation of mature oocytes on an elective basis for the purpose of delayed childbearing. As of 2013, the American Society for Reproductive Medicine (ASRM) no longer considers egg freezing experimental, and large organizations such as Apple, Facebook, and the US military have started offering social egg freezing as an employee benefit.[10-13]

Why is egg freezing needed?
Over the past 30 years the industrialized world has seen a dramatic increase in the age of first birth.[14,15] According to Statistics Canada, 2010 marked the first time in our history that more women in their 30s were having children than women in their 20s.16 In 2011, there were 52.3 babies born per 1000 women age 35 to 39, compared with 45.7 per 1000 women age 20 to 24.[17] In British Columbia, the percentage of live births to women age 35 and older rose from 11% in 1990 to 23% in 2011, while births in the 20 to 34 age category fell from 83% to 74% over the same period.[18] In one study, the lack of a partner was by far the most common reason women gave for not pursuing childbearing earlier, followed distantly by professional and financial reasons.[15] When researchers from the University of British Columbia surveyed 360 female undergraduate students about their childbearing intentions, they found that “although most women were aware that fertility declines with age, they significantly overestimated the chance of pregnancy at all ages and were not conscious of the steep rate of fertility decline.”[19] It is therefore incumbent on health care providers to educate women about the risks of advancing reproductive age and to ensure that patients are not “sleepwalking” into unintended childlessness.[20] Women need to be made aware of the three main risks of delayed childbearing: infertility, aneuploidy, and miscarriage.

Egg freezing is currently the best way to provide females with reproductive potential more like that of males. The process of spermatogenesis takes approximately 70 days and continues throughout a man’s life, allowing most men to maintain fertility in perpetuity.[21] A woman, in contrast, is born with a finite number of eggs that diminish over 5 or 6 decades until menopause is attained. In a unique study of 122 necropsy and surgical specimens from females age 0 to 51 years, Hansen and colleagues used stereology techniques to count the number of nongrowing follicles present in the ovary and establish each subject’s ovarian reserve.[22] At 20 weeks’ gestation, we know a female fetus has her lifetime maximum of 6 to 7 million oogonia.[21] Hansen and colleagues found that a newborn’s ovaries contained approximately 1 million eggs, and by puberty this number had fallen by half.[22] The most clinically remarkable decline was seen between the 30 to 34 age group, where 200 000 eggs remained, and the 35 to 39 age group, where the average was less than 40 000. The menopause threshold was reached at approximately 51 years, when fewer than 1000 nongrowing follicles were seen. Importantly, menstruation is often erroneously equated with fertility, when in fact most women lose the ability to conceive 10 years or more before they cease menstruating.[23] As well, external gonadotoxic factors such as chemotherapy, pelvic radiation, ovarian surgery, and autoimmune disorders can have an impact on ovarian reserve by accelerating the normal age-related loss of eggs.[21]

Aneuploidy, or chromosomal error, is more common in embryos derived from older eggs. This is believed to result from the age-related deterioration in cellular mechanisms required for meiotic spindle function.[21] When meiosis is reinitiated by the luteinizing hormone (LH) surge or fertilizing sperm, chromosome or sister chromatid segregation can occur unevenly between the egg and its resulting polar bodies.[21] Newly developed techniques permit the analysis of embryos created through in vitro fertilization (IVF) and the selection and transfer of only those embryos that are euploid in a process termed preimplantation genetic screening (PGS) or comprehensive chromosome screening (CCS). The embryo’s trophectoderm (the outer layer destined to form the placenta) is biopsied on day 5 or 6 after fertilization and the chromosomal complement of those cells is assessed. In a large series of 15 169 trophectoderm biopsies from 2701 patients, only 20.7% of embryos from women age 29 were abnormal, compared with 34.5% of embryos from women age 35 and 58.2% from women age 40. By age 43, only 16.6% of embryos were chromosomally normal.[24]

Knowing that a substantial majority of conceptions in women over 40 are aneuploid helps us understand why there is an increase in the time needed to achieve pregnancy and in pregnancy losses, both clinical and biochemical, as women age. Miscarriage rates in natural pregnancies for women younger than 30 are 7% to 15% and become marginally higher for women 30 to 34 at 8% to 21%. In the 35 to 39 age group the rate is 17% to 28% and in the over 40 age group 34% to 52% of pregnancies end in miscarriage.[21]

In short, women in our society are waiting longer to start families but their ovaries remain programmed for reproduction at a younger age. As women continue to strive for equality, they are finding it increasingly difficult to reconcile their professional and political goals with the rigid biology of reproduction. In the words of one researcher, egg freezing may be able to “bridge the gap between reproductive prime and when a woman is realistically ‘ready’ to have children.”[15]

What does egg freezing involve?
Women seeking social egg freezing at a fertility clinic begin by having their ovarian reserve assessed. This is done with blood tests for early follicular phase (day 3) follicle-stimulating hormone (FSH) and antimüllerian hormone (AMH) levels, and then an antral follicle count (AFC) performed by transvaginal ultrasound. Findings from these investigations provide an estimate of the patient’s remaining eggs compared with other women her age. They also help the fertility doctor determine how many eggs can safely and/or realistically be obtained in one cycle of controlled ovarian stimulation.

In vitro fertilization involves an average of 10 days of gonadotropin injections (FSH and LH) before the eggs are prompted to undergo maturation by triggering an endogenous LH surge, or by the administration of human chorionic gonadotropin (hCG). Egg retrieval is an outpatient surgical procedure that takes approximately 10 minutes under conscious sedation. A transvaginal ultrasound-guided needle is used to aspirate the fluid from each ovarian follicle, which an embryologist then examines to isolate eggs. After that, mature eggs are counted and prepared for freezing.

As the largest cells in the human body, eggs are very difficult to freeze. Their spherical shape, high water content, and low surface area to volume ratio make eggs especially difficult to permeate with cryoprotectants and prone to intracellular ice crystal formation.[25] Eggs are susceptible to meiotic spindle and lipid membrane damage, hardening of the zona pellucida, and depolymerization of microtubules.[25] For years the slow-freezing technique used for embryos was used for eggs, but with little success. In 2004, when changes to Italian reproduction laws made it illegal to freeze embryos, the need to cryopreserve supernumerary eggs[4] sparked the development of a new, more efficient flash-freezing method called vitrification.[25,26] This technique uses ultra-rapid cooling rates and high concentrations of cryoprotectants. There is now a growing body of evidence that IVF outcomes with vitrified eggs are the same as those with fresh eggs.[6,27-29]

How well does egg freezing work?
Most of the evidence on egg freezing comes from research in countries where remuneration for human gametes is legal, such as the US and Spain.[5] In order to ease the logistical burden of anonymous egg donation in synchronous IVF cycles, fertility clinics will often bank frozen eggs from donors and subsequently distribute them to recipients for warming and fertilization at a more convenient time. In a review of 3610 donor egg warming procedures, the vitrified egg survival rate was 90.4% and the oocyte-to-baby rate was 6.5%.[30] Since these eggs came from donors who were typically in their 20s and selected for a good prognosis, it may be too optimistic to extrapolate these findings to social egg freezing. Given that the widespread utilization of vitrification and the nonexperimental use of egg freezing have increased only recently, long-term outcome data from large trials of social egg freezing are still forthcoming.

In 2016, reports were published for two studies that examined large numbers of elective egg freezing cycles. The first study, by Cobo and colleagues,[27] included 1468 women who underwent social egg freezing. The researchers found that 137 of these subjects (9.3%) had returned to use their eggs, that the average age of those returning was 37.7 years, and that the overall egg survival rate was 85.2%. For women who were 35 or younger at the time of egg freezing and who banked 10 eggs, the average live birth rate when 10 eggs were used was an impressive 60.5% (95% CI, 34.5-89.5%). In the group of women 36 or older, the same 10 eggs yielded a significantly lower live birth rate of 29.7% (95% CI, 15.2%-34.2%).[27] The second study, by Doyle and colleagues,[28] reported on 1283 vitrified eggs from 128 autologous IVF cycles. The researchers found an egg-to-live-birth efficiency rate of 6.4% for these autologous cycles, which was very similar to the rate of 6.5% reported previously for donor cycles.[30] When they compared the performance of autologous frozen eggs to fresh eggs in IVF cycles, the overall live birth rate was reassuringly similar (39% versus 35%).[28] To put these figures in perspective, it helps to consider what success rate a woman of a given age could expect if she were to pursue an IVF pregnancy today rather than freezing her eggs. Aggregate data from 31 IVF clinics across Canada in 2014 found the following average clinical pregnancy rates for each age group: younger than 35 years = 48.7%, 35 to 37 = 43.5%, 38 to 40 = 34.3%, 41 to 42 = 20.5%, 43 or older = 14.4%.[31] This means that for women younger than 36, social egg freezing appears to be at least as good as the national IVF averages. Given that social egg freezing is relatively new, the vast majority of eggs retrieved for elective freezing so far are presently in cryostorage. At our centre, two patients have returned to use their eggs, both of whom have ongoing pregnancies at the time of publication.

Egg vitrification is gaining acceptance with the improving efficiency of our freezing techniques.[5,9] The best chance for a future pregnancy appears to result from freezing at least 8 to 10 eggs before age 36.[27] In Canada, the average cost of an egg freezing cycle, including procedures and medication, ranges from $7000 to $10 000 and annual storage fees range from $200 to $400. To determine the most cost-effective approach, a US study developed a decision-tree model that looked at the cost of egg freezing compared with taking no action between age 25 and 40 and assuming an attempt at procreation was made 3, 5, or 7 years after the initial decision.[14] The researchers weighed the chances of spontaneous pregnancy against the chances of success with IVF using frozen eggs in various age groups. They concluded that although the highest probability of live birth was achieved when egg freezing was performed before age 34 (greater than 74%), egg freezing was most cost effective at age 37.[14]

What are some concerns about egg freezing?
Egg freezing does not appear to increase the likelihood of fetal malformations or pregnancy complications. Initial reports from the Italian experience up until 2007 and a case series of 900 infants published in 2009 both showed no increase in congenital anomalies among children resulting from egg freezing.[32,33] A more recent study compared 804 pregnancies from vitrified eggs with 996 pregnancies from fresh eggs and found no difference in adverse obstetrical outcomes, including gestational diabetes, hypertension, preterm birth, cholestasis, and anemia.[34] There were also no differences in perinatal outcomes, including gestational age at delivery, preterm birth, APGAR scores, birth defects, neonatal ICU admission, and perinatal mortality.[34]

Vitrified eggs can likely remain in cryostorage indefinitely, although many clinics impose an upper patient age limit of 50 years for embryo transfer. Evidence from donor egg IVF cycles has demonstrated that the uterus is capable of sustaining a pregnancy well beyond menopause with the administration of exogenous estrogen and progesterone.[35] The cardiovascular and other physical demands of pregnancy present more substantial challenges, as do the ethical implications of postmenopausal motherhood.[36,37] Despite the encouraging statistics, media attention, and corporate support for egg freezing, the practice has generated much controversy. Some of the more frequently cited concerns are the financial barriers and the lack of access for all women, along with the false sense of security that the technology may provide. Women face societal pressures to achieve their education and professional goals, to meet the right partner, and to start a family within a relatively short 10- or 15-year period. Egg freezing may be seen by some of these women as a way to “have it all” or for employers to “expect it all.”[5,37] Ethicists have stated that health care providers should “frame discussions” about egg freezing “within the broader context of reproductive health and family-making to assist women in making informed choices.”[37]

Conclusions
Social egg freezing is a safe and viable option for women in our society. It does not provide women with the same reproductive longevity that men enjoy, but it can allow women to delay childbearing for 2 to 10 years and may be a reasonable choice for women wishing to do this. Egg freezing appears to be most effective when at least 8 to 10 eggs are banked before age 36 and most cost-effective when undertaken at age 37. Women may regard social egg freezing as a reproductive insurance policy or simply as a backup plan. However, it is important for women to understand that no number of stored eggs will guarantee a baby in the future.

Women are already choosing to have children later in life. Whether egg freezing increases or decreases the pressure on women to make choices about their partners, professional goals, and fertility lies in the eye of the beholder. It is important for health care providers to educate women about the risks of infertility, aneuploidy, and miscarriage that come with delaying the creation of a family. Women have the right to shift priorities as they adapt to their evolving roles in society, and social egg freezing may help them do this.

Competing interests
None declared.

This article has been peer reviewed.

References

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3. Roberts J, Ronn R, Tallon N, Holzer H. Fertility preservation in reproductive-age women facing gonadotoxic treatments. Curr Oncol 201522:e294-e304.
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5. Lockwood G, Johnson MH. Having it all? Where are we with “social” egg freezing today? Reprod Biomed Online 201531:126-127.
6. Cobo A, Kuwayama M, Perez S, et al. Comparison of concomitant outcome achieved with fresh and cryopreserved donor oocytes vitrified by the Cryotop method. Fertil Steril 200889:1657-1664.
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10. Practice Committees of American Society for Reproductive Medicine Society for Assisted Reproductive Technology. Mature oocyte cryopreservation: A guideline. Fertil Steril 201399:37-43.
11. Practice Committee of American Society for Reproductive Medicine. Ovarian tissue cryopreservation: A committee opinion. Fertil Steril 2014101:1237-1243.
12. Tran M. Apple and Facebook offer to freeze eggs for female employees. The Guardian. 15 October 2014. Accessed 17 June 2016. www.theguardian.com/technology/2014/oct/15/apple-facebook-offer-freeze-e. .
13. Lampert N. New fertility options for female soldiers. The Atlantic. 29 February 2016. Accessed 17 June 2016. www.theatlantic.com/health/archive/2016/02/fertility-women-soldiers/471537/.
14. Mesen TB, Mesereau JE, Kane JB, Steiner AZ. Optimal timing for elective egg freezing. Fertil Steril 2015103:1551-1556.
15. Hodes-Wertz B, Druckenmiller S, Smith M, Noyes N. What do reproductive-age women who undergo oocyte cryopreservation think about the process as a means to preserve fertility? Fertil Steril 2013100:1343-1349.
16. Cohn D. In Canada, most babies now born to women 30 and older. Pew Research Center. 10 July 2013. Accessed 27 September 2016. www.pewresearch.org/fact-tank/2013/07/10/in-canada-most-babies-now-born-. .
17. Statistics Canada. Report on the demographic situation in Canada, 2008 to 2012. Released 7 September 2013. Accessed 27 September 2016. www.statcan.gc.ca/daily-quotidien/130709/dq130709a-eng.htm.
18. British Columbia Vital Statistics Agency. Annual report 2011. Accessed 17 June 2016. www2.gov.bc.ca/gov/content/life-events/statistics-reports/annual-reports/2011.
19. Bretherick KL, Fairbrother N, Avila L, et al. Fertility and aging: Do reproductive-aged Canadian women know what they need to know? Fertil Steril 201093:2162-2168.
20. Lemoine M-E, Ravitsky V. Sleepwalking into infertility: The need for a public health approach toward advanced maternal age. Am J Bioeth 201515:37-48.
21. Fritz MA, Speroff L. Clinical gynecologic endocrinology and infertility. 8th ed. Philadelphia: Lippincott Williams & Wilkins 2011.
22. Hansen KR, Knowlton NS, Thyer AC, et al. A new model of reproductive aging: The decline in ovarian non-growing follicle number from birth to menopause. Hum Reprod 200823:699-708.
23. Schattman GL. A healthy dose of reality for the egg-freezing party. Fertil Steril 2016105:307.
24. Franasiak JM, Forman EJ, Hong KH, et al. The nature of aneuploidy with increasing age of the female partner: A review of 15,169 consecutive trophectoderm biopsies evaluated with comprehensive chromosomal screening. Fertil Steril 2014101:656-663.
25. Kuwayama M. The human oocyte: Vitrification. In: Gardner DK, Weissman A, Howles CM, Shoham Z, editors. Textbook of assisted reproductive techniques. 4th ed. Boca Raton, FL: Taylor & Francis Group 2012.
26. Kuwayama M. Highly efficient vitrification for cryopreservation of human oocytes and embryos: The Cryotop method. Theriogenology 200767:73-80.
27. Cobo A, Garcia-Velasco JA, Coello A, et al. Oocytes vitrification as an efficient option for elective fertility preservation. Fertil Steril 2016105:755-764.
28. Doyle JO, Richter KS, Lim J, et al. Successful elective and medically indicated oocyte vitrification and warming for autologous in vitro fertilization, with predicted birth probabilities for fertility preservation according to number of cryopreserved oocytes and age at retrieval. Fertil Steril 2016105:459-466.
29. Potdar N, Gelbaya TA, Nardo LG. Oocyte vitrification in the 21st century and post-warming fertility outcomes: A systematic review and meta-analysis. Reprod Bio-med Online 201429:159-176.
30. Cobo A, Garrido N, Pellicer A, Remohi J. Six years’ experience in ovum donation using vitrified oocytes: Report of cumulative outcomes, impact of storage time, and development of a predictive model for oocyte survival rate. Fertil Steril 2015104:1426-1434.
31. Canadian Fertility Andrology Society. Canadian Assisted Reproductive Technologies Register (CARTR) Plus 2014 report. Accessed 27 September 2016. https://cfas.ca/cartr-annual-reports/.
32. Borini A, Bianchi V, Bonu MA, et al. Evidence-based clinical outcome of oocyte slow cooling. Reprod Biomed Online 200715:175-181.
33. Noyes N, Porcu E, Borini A. Over 900 oocyte cryopreservation babies born with no apparent increase in congenital anomalies. Reprod Biomed Online 200918:769-776.
34. Cobo A, Serra V, Garrido N, et al. Obstetric and perinatal outcome of babies born from vitrified oocytes. Fertil Steril 2014102:1006-1015.
35. Centers for Disease Control and Prevention. Division of Reproductive Health. Assisted reproduction report, 2012. Accessed 23 February 2016. www.cdc.gov/art/pdf/2012-report/art_2012_graphs_and_charts_final.pdf.
36. Kimberly L, Case A, Cheung AP, et al. Advanced reproductive age and fertility: no. 269, November 2011. Int J Gynaecol Obstet 2012117:95-102.
37. Petropanagos A, Cattapan A, Baylis F, Leader A. Social egg freezing: Risk, benefits and other considerations. CMAJ 2015187:666-669.

Dr Dunne is a clinical assistant professor in the Department of Obstetrics and Gynaecology at the University of British Columbia and a co-director of the Pacific Centre for Reproductive Medicine. Dr Roberts is a clinical assistant professor in the Department of Obstetrics and Gynaecology at the University of British Columbia and a co-founder/director of the Pacific Centre for Reproductive Medicine. He is the 2016–17 president of the Canadian Fertility and Andrology Society.


4 DISCUSSION

In this study, we have provided new insights into the effects of CSE on the regulatory mechanisms of angiogenesis and decidualization in ESCs. CSE induced VEGF mRNA expression levels in a dose-dependent manner, as well as HIF-1α protein levels in ESCs. These results suggest that HIF-1α activation by CSE enhances VEGF mRNA expression in ESCs. Additionally, to the best of our knowledge, our study is the first to demonstrate that CSE induces differential effects on PRL and the other decidual specific factors evaluated depending on the concentration of CSE tested. In brief, low concentrations of CSE increased the expression levels of PRL and other decidual specific factors, whereas high concentrations of CSE suppressed the expression levels. Based on our findings, CSE affects the production of angiogenic factors, HIF-1α activation, and decidualization in ESCs.

The toxicity test for cigarette smoke is commonly used in animal or cell experiments, and CSE is widely employed in in vitro models. 28 Earlier studies report that 1% CSE approximately corresponds to exposures associated with smoking slightly less than two packs of cigarettes per day in pulmonary artery endothelial cells. 29, 30 Based on these studies, we propose that 0.25% CSE concentration closely represents exposure faced by average smokers. However, whether CSE concentration is similar between pulmonary artery endothelial cells, which directly absorb cigarette smoke, and ESCs is unknown.

Concurring with our results, nicotine in cigarette smoke has been shown to induce VEGF expression in human ESCs, regardless of ovarian steroid hormones. 31 VEGF is essential for implantation and placentation 32 however, higher levels of VEGF may disrupt normal angiogenesis through an overstimulation of blood vessels leading to disturbed vascular architecture. 33, 34 Taken together, it is important to consider the influence of these results of the present study on implantation, as the effects of CSE on angiogenesis may adversely affect the establishment of pregnancy.

In the promoters encoding VEGF genes, HIF-1α has been shown to directly bind to the hypoxia response element. In the human endometrium, HIF-1α is expressed with increasing intensity from the premenstrual to the menstrual phase. 35 Previous studies showed that both HIF-1α and HIF-2α have a functional role in embryo implantation. 36, 37 HIF-1α is a transcription factor known to play a critical role in the cellular response to hypoxia. However, under normoxic conditions, HIF-1α is synthesized in a kinase inhibitor-sensitive manner via PI3K, Akt, and mTOR pathways. 38, 39 In fact, CSE is reported to activate HIF-1α under normoxic conditions both in vitro and in vivo. 40 In human lung adenocarcinoma A549 cells, CSE induced the expression of VEGF via HIF-1 activation in a reactive oxygen species (ROS)-dependent manner. 40 HIF-1α also is shown to be involved in the induction of VEGF in human ESCs and directly binds to the promoters of the genes encoding VEGF. 41 Our results concur with findings from earlier studies that showed CSE-mediated induction of VEGF expression and HIF-1α accumulation in ESCs under normoxia conditions in the absence of E2 and MPA.

GLUT1 has a HIF-1α binding sequence in its promoter. 27 In this study, CSE simultaneously increased GLUT1 mRNA expression and the accumulation of HIF-1α. We recently demonstrated that echinomycin, a small molecule inhibitor of HIF-1α activity, substantially reduced GLUT1 expression under hypoxia in ESCs, suggesting that HIF-1α plays a major role in regulating GLUT1 expression. The physiological role of GLUT1 is that in a hypoxic environment such as menstrual and implantation periods, ESCs increase extracellular glucose uptake and enhance glycolysis, thereby obtaining energy. 42 Herein, these findings suggest that CSE-induced HIF-1α plays a key role in the regulation of GLUT1 and VEGF expression.

The observation from this study that 0.25% CSE significantly decreased ANGPT1 expression, but had no effect on ANPGT2 expression, is consistent with previous data showing that hypoxia reduced the mRNA expression and protein production of ANGPT1 in ESCs, whereas those of ANGPT2 remained unaffected. 9 Recent studies have reported that ROS decreases the expression of ANGPT1 mRNA and proteins in cultured human ESCs. 43 CSE-induced ROS may play a role in the reduction of ANGPT1 expression. Interestingly, VEGF expression in ESCs is affected by low concentrations of CSE, whereas ANGPT1 expression is modulated only at 0.25% CSE concentration. Further research is needed to clarify the different mechanisms that regulate VEGF and ANGPT1 expression in response to CSE.

We demonstrated that CSE affects the expression of decidual specific factors. The levels of PRL mRNA and PRL protein were elevated after treatment with 0.01% and 0.025% CSE compared to control, but expression levels were then suppressed at 0.1% and 0.25% CSE concentrations in the presence of E2 and MPA. However, without E2 and MPA, CSE had no effect on the levels of PRL expression, suggesting that CSE affects PRL levels during decidualization. The observed morphological changes correlated with the changes in PRL levels after exposure to different CSE concentrations. Cadmium, one of the major contaminants of cigarette smoke, markedly elevates PRL levels and stimulates decidualization in ESCs. 44 Another study also showed that CSE increased the expression of endometrial homeobox 10 and the progesterone receptor and promoted early decidualization in immortalized endometrial cell lines of ESC. 45 In this study, treatment with 0.01% and 0.025% CSE also upregulated IGFBP-1 mRNA in ESCs. The time point of IGFBP-1 secretion, which is about 10 d after the luteinizing hormone peak in vivo, is relevant for a marked reduction in endometrial receptivity and a rapidly increasing risk of implantation failure. 8 Therefore, CSE may affect the endometrial receptivity by upregulating IGFBP-1.

There are very few reports that CSE suppresses decidualization. To the best of our knowledge, there is one report using female rat models showing the potential effects of nicotine on endometrial decidualization by assessing by the weight of the uterus after mechanically induced decidualization. The authors concluded that there was an adverse effect on the decidualization process resulting in a lower uterus weight after nicotine administration. 46 In the present study, we showed that CSE resulted in differential gene expressions depending on the concentration tested. This may be attributed to concentration differences of the principal substances within CSE, at the different concentrations of the CSE extract solution. Further studies are warranted to determine the influence of the main individual components of CSE on gene expressions.

As previously mentioned, HAND2 is a transcription factor and progestin-induced HAND2 plays a key role in the regulation of PRL and IGFBP-1 expression in ESCs. 47 It is also reported that HAND2 enhanced IL-15 and suppressed FGF9 in ESCs. 17, 19 Our study demonstrates that CSE may exert effects on the expression of PRL, IGFBP-1, and IL-15 via HAND2. In contrast, E2 and MPA attenuated the FGF9 mRNA levels, but CSE treatment alone did not affect expression.

There are several limitations to our study that need to be considered. First, the effects of CSE on the mRNA levels of the decidual specific factors were measured however, future studies need to confirm the translation of the mRNA into protein. Additionally, all patients from whom the tissue samples were obtained were non-smokers. Thus, further evaluation of the effects of CSE on ESCs from non-smokers and smokers is warranted. Moreover, the impact of smoking on endometrial maturation including angiogenesis and decidualization needs to be corroborated in vivo. Another limitation of this study is the low sample number. Increasing the sample number could provide stronger statistical verification of our findings. The final limitation is the concentration and composition of CSE. As mentioned above, previous studies have reported the concentrations of CSE in smokers, 27, 28 but these findings may or may not be applicable to our study, and hence, further studies on the tissue concentrations of CSE in ESCs are needed. Moreover, cigarette smoke contains more than 4000 toxic compounds, 48 of which one or more can induce or reduce the expression of genes that play a role in angiogenesis and decidualization of ESCs. Isolation and characterization of these compounds may help clarify the paradoxical observation that CSE caused differential gene expressions depending on the concentration tested and also lead to the development of therapeutic strategies.

Collectively, our study demonstrates that CSE above 0.025% enhances the expression of angiogenic factors, and CSE above 0.01% affects the expression of decidual specific factors in ESCs in the presence of E2 and MPA. These results highlight that exposure to even a small amount of cigarette smoke could affect ESCs. Our study provides a novel insight in that cigarette smoke directly affects the human endometrium. Moreover, our results support epidemiological studies that cigarette smoke has an adverse effect on reproductive outcome.


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