Compositions methods and devices for embryo implantation for in vitro fertilization

The present invention is directed to compositions and apparatus to be used in methods of implantation of embryos for in vitro fertilization. The methods include insertion of embryos that have attached adhesives. Such methods, compositions and apparatus can be used with any mammals, including humans, farm animals and exotic or endangered animals.

TECHNICAL FIELD
 The present invention relates to compositions, methods and devices for in
 vitro fertilization. More particularly, the present invention relates to
 compositions, methods and devices to aid in implantation of embryos.
 BACKGROUND OF THE INVENTION
 Treatment of infertility problems is a growing area of health care.
 Approximately 50,000 human in vitro fertilization (IVF) procedures are
 performed in the US annually. Although costs vary widely depending on
 drugs, testing and other laboratory fees, typical IVF charges are on the
 order of $10,000 per procedure, not counting travel, lost work time, and
 emotional costs for the patients. Additionally, in treatment of animals,
 the ability to control the birth of offspring is important for
 agricultural concerns and for preservation of endangered species.
 Many approaches are being developed to resolve problems with infertility.
 Infertility is understood to be the inability to conceive after six to
 twelve months of sexual activity without the use of contraceptives,
 depending on the age of the persons involved. Because infertility exerts
 extreme physical, emotional and financial stresses on those who are unable
 to conceive, there is a great need for improved aids for reproduction.
 These aids are typically referred to as Assisted Reproductive Technologies
 (ART). By far the most common ART component is IVF, which has grown
 explosively in the two decades since it was developed. In its simplest
 form, IVF consists of pharmaceutical stimulation of the female's ovaries
 to produce a large number of follicles. Eggs surgically harvested from
 these follicles are then mixed in the laboratory with the male's sperm. If
 fertilization is successful, the embryos are incubated for a short time
 and then transferred back to the female. If one of these embryos implants
 in the uterine wall, a successful pregnancy may follow.
 There are several modifications of this basic technique. For example,
 intracytoplasmic sperm injection (ICSI) can be used for cases of low sperm
 count or cases where the sperm has difficulty fertilizing the egg. Another
 IVF modification is Assisted Hatching (AH), a procedure in which the zona
 pellucida (the outer wall of the embryo) is mechanically cut or chemically
 etched, thereby partially exposing the embryo. In some laboratories, this
 procedure significantly improves implantation rates, particularly for
 older patients. Finally, IVF procedures can also incorporate donor
 tissues, including sperm, ova and embryos, for those individuals who
 cannot produce their own.
 Despite its great successes, IVF has several significant problems. First
 and foremost, the procedure is unpredictable. Although the ideal result of
 any IVF procedure is a single, live birth, a viable pregnancy occurs in
 only about 30% of all procedures. Conversely, IVF may result in a
 pregnancy with multiple embryos. In this regard, twins and triplets pose
 relatively few risks beyond a single embryo pregnancy. The potential for
 problems, however, increases for higher order births. Selective embryo
 reduction is therefore often recommended for these cases which increases
 the psychological trauma for the parents.
 Like unassisted reproduction, IVF begins with a source of sperm and ova.
 There is a virtually 100% certainty of obtaining these materials, using
 donor tissues if necessary. Next, fertilization occurs, and good IVF
 laboratories typically have a fertilization success rate of about 75%,
 using ICSI if appropriate. After a short incubation period, the resulting
 embryo is then introduced into the uterus, where implantation occurs.
 Implantation is generally the limiting factor in overall IVF success.
 Implantation itself, however, consists of several steps. First, the embryo
 must enter the uterine cavity. In normal reproduction, without ART, the
 embryo descends through the fallopian tubes. The embryo then comes into
 contact with some point on the uterine wall. Next, the embryo and wall
 surfaces fuse at the contact point. The uterine wall properties then
 change dramatically at the implantation site, thus allowing the embryo to
 become fully implanted. For IVF, the embryo is carried into the uterine
 cavity in a solution injected from a syringe inserted through the cervical
 canal.
 Although the implantation process appears to be simple enough, it is
 actually quite complicated and requires the coordination of many factors,
 some of which are unknown. A failure of any one of these processes
 prevents implantation and thus pregnancy. Furthermore, it is believed that
 implantation failures may indeed be the reason that only about 20% of even
 the most fertile couples conceive in a given month of attempting
 pregnancy.
 Improving the implantation rate would make the IVF process more reliable,
 more effective and yield many benefits. Improving the effectiveness of the
 IVF process above its current 30% would reduce the need for repeat
 procedures, a critical factor considering the costs and stresses involved
 for the patients. Another benefit is that an improved success rate would
 make better use of the quite limited supply of donor ova and embryos.
 Improving the success rate would also further extend the supply of donor
 tissues by enabling some patients, particularly those who are older but
 otherwise healthy, to use their own tissues. Such patients would be more
 than willing to leave the donor program given the opportunity to have
 children of their own genetic basis. Yet another benefit is that by
 reducing the number of embryos required for a successful procedure, the
 incentive to overstimulate the ovaries is reduced, thereby reducing
 possible harmful side effects to the patient. Finally, improved
 implantation rates would eliminate the incentive to transfer large numbers
 of embryos back to the uterus, thereby preventing multiple births.
 These advantages have been noted before, resulting in a variety of efforts
 to improve the implantation process, including both chemical and
 mechanical methods. The chemical techniques employ both natural and
 artificial compounds to improve the conditions of the embryo, the uterus,
 or both.
 Many of these chemical efforts employ pharmaceutical modification of the
 uterine surface to make it more receptive to the embryo. Although these
 efforts are promising, there is concern that any agent strong enough to
 alter the properties of the uterus may also harm the embryo, thereby
 leading to birth defects. Another approach to improve implantation
 involves incubating the embryos to the blastocyst or later stages before
 transfer. Although this approach is promising, one European group has
 recently established an Internet registry to track the possibility of
 related birth defects. At the present time, none of these methods are
 effective in increasing the success at a viable pregnancy.
 Mechanical means have focused on assisted hatching (AH) and tissue
 cultures. AH involves eroding the wall of the embryo by chemical and/or
 physical attack so that the embryo can expand and attach more readily, a
 technique particularly useful for patients over 38 years of age. Tissue
 cultures of uterine wall cells promise a more hospitable environment while
 still in vitro, with the hope that these conditions will prevail after
 transfer.
 None of these techniques has produced much success. Thus, what is needed
 are compositions, methods and devices to aid in embryo transfer and
 implantation for in vitro fertilization.
 SUMMARY OF THE INVENTION
 The present invention relates to compositions, methods and devices for
 increasing the success of implantation of an embryo for in vitro
 fertilization. More particularly, the present invention relates to
 compositions, methods and devices for attaching an embryo to a surface
 that is then placed within the uterine cavity. Such compositions, methods
 and devices are contemplated for use in any animal system, including
 humans, and animal husbandry, such as cattle, sheep and swine, and for
 exotic animals. The following compositions and methods are intended as
 means to enhance the implantation rates now observed.
 IVF is a complicated, sophisticated process. The present invention is
 directed to improving implantation rates by providing compositions and
 devices in methods for implantation. One immediate result of improved
 implantation rates is fewer total IVF procedures, with psychological,
 physical, and financial savings to the patient. Another immediate result
 is fewer embryos transferred at any given time, leaving more embryos for
 later transfer without requiring further retrieval. The present invention
 thus also reduces that likelihood of multiple births.
 A preferred embodiment of the present invention is a combination of
 mechanical manipulation of the embryo with compositions capable of
 supporting and containing the embryo, or compositions and mechanical
 manipulations capable of enhancing the implantation of the embryo. The
 devices of the present invention can be used with any such advances to
 achieve ideal IVF success.
 Accordingly, it is an object of the present invention to provide methods to
 aid in implantation for in vitro fertilization.
 Another object of the present invention to provide compositions to aid in
 implantation for in vitro fertilization.
 It is yet another object of the present invention to provide devices for
 aids in implantation for in vitro fertilization.
 It is another object of the present invention to provide compositions,
 methods and devices that allow for the formation of a structure that holds
 one or more embryos in a stable form that is placed within the body to aid
 in implantation.
 It is a further object of the present invention to increase the success
 rate of in vitro fertilization by aiding in the implantation of embryos.

DETAILED DESCRIPTION
 The present invention comprises compositions, methods and devices to aid in
 implantation of embryos. These compositions, methods and devices are
 particularly advantageous for in vitro fertilization. Such methods and
 devices may be used for humans, but also may be used for animals or any
 other system where embryo implantation is used. As used herein, embryo
 includes a fertilized egg or other structure that is capable of developing
 into a new individual.
 Most of the current IVF treatments are directed to changes in the uterine
 wall. For example, progesterone supplements are commonly prescribed to
 avoid the problems associated with luteal phase defects. Likewise, to
 improve the success rate of IVF procedures, biological markers in the
 uterine surface can be used passively for timing purposes, or the surface
 itself can be altered actively by pharmaceutical agents. Although these
 and similar approaches show significant promise, they nevertheless have
 the potential problem of inducing serious birth defects.
 The work to date involving embryos has emphasized donor tissues, instead of
 pharmaceutical approaches. Specifically, it has been found that women in
 their later thirties and early forties using donor eggs have essentially
 the same fertility rate as the younger donors. A similar result holds for
 donor embryos, which is not surprising because sperm function changes
 relatively little over the normal paternal age range, leaving the
 effective age of the embryo to be that of the egg. With such advances in
 ART, even post-menopausal women can give birth, using appropriate hormone
 therapy. Preliminary work is currently under way at several IVF
 laboratories to remove most of the genetic material from the center of a
 donor egg and replace it with the older patient's material. The desired
 result is that the patient would have an egg of essentially her own
 genetic basis, while retaining the higher likelihood of pregnancy of the
 younger donor. Although no births have been reported to date, this
 technique does show significant promise.
 Though not wishing to be bound by any particular theory, it is theorized
 that implantation depends on the uterine cavity being in contact with the
 embryo for a sufficient amount of time so that the necessary bonds can
 form. In particular, only one site of the uterine wall needs to be in
 contact with only one site of the embryo.
 Of course, in nature this does not always happen. Instead, the embryo
 essentially bounces from site to site until a suitable site can be found.
 For in vitro processes, this situation is even worse, with the embryo
 floating in the suspension fluid before any attachment can be made. The
 time spent by the embryo by floating in the suspension fluid may account
 for some of the lower observed implantation rates for such processes.
 Any preliminary attachment by the embryo is weak and extends only over a
 limited area, and bond rupture can occur readily, again leaving the embryo
 unattached. Such ruptures would be expected to be of particular concern
 for older patients because the tougher shell of the embryo would be less
 prone to form strong bonds quickly. Repeated ruptures thus eventually
 result in implantation failure, as often observed for older patients using
 their own eggs without AH. Conversely, the use of AH or donor eggs leads
 to stronger bonds, in agreement with the enhanced implantation rates
 observed for these approaches.
 This bonding requirement holds for all stages of embryo development prior
 to transfer, and the present invention can be used with blastocyst and all
 other alternative stage approaches. Likewise, the present invention can
 also be used with chemical treatments of the uterine wall or cavity that
 are known to those skilled in the art. Safe and effective pharmaceutical
 and/or advanced incubation processes can be used in a combination of these
 procedures with the present invention.
 Though not wishing to be bound by any particular theory, the following
 theory of implantation is suggested. Mechanically, the limiting step in
 implantation is the first contact of the embryo with the uterine wall. At
 this juncture, the embryo has only a minimal contact area with the wall
 surface, compared to the much greater contact area that develops as the
 uterine wall eventually envelopes the embryo. Also at first contact, the
 bonds between the embryo and the wall are only just beginning to form,
 compared to the much stronger bonds that form as implantation proceeds.
 The net effect is that implantation begins with weak bonds joining only a
 small area of the embryo to the uterine wall, progressing to strong bonds
 over virtually the entire embryo surface.
 As a direct clinical consequence of the progression of binding in
 implantation, IVF patients are advised to refrain from sexual intercourse,
 heavy lifting, or any other vigorous physical activity for at least two
 days after transfer. Such higher levels of activity can be safely
 tolerated later in the pregnancy after more secure attachment has been
 achieved by the embryo.
 It is an object of the present invention to hold the embryo in continuous
 contact with a selected point of the uterus, and to enhance the bonding
 between the embryo and the uterine wall, thereby aiding the implantation
 of the embryo. Thus, the present invention includes, but is not limited
 to, embryo preparation plates, biological adhesives, and applicators.
 An element of the present invention is a device for holding the embryo for
 further manipulations, the embryo preparation plate. The present invention
 contemplates a device that is capable of stabilizing the embryo so that
 the embryo can be manipulated in the manner of the methods of the present
 invention. No particular design is required by the present invention, and
 any device that holds the embryo without damage, and allows for the
 application of adhesives or other manipulations is contemplated by the
 present invention. A preferred device is a modification of a flat
 incubation plate so that the plate has hemispheric depressions the size of
 the embryo, including floatation space. Another embodiment is a flat
 plastic plate, containing a hemispherical depression with rounded edges.
 An inexpensive method for making such a plate comprises forcing a normal
 straight pin into the soft plastic of the plate, yielding a conical
 depression. An acid etch then removes the sharp edges to create the
 desired shape. For commercially produced devices, micromachining
 techniques are used to produce a high quality mold having the desired
 shape, which would then be used for mass production of plates with
 hemispheric depressions.
 Other elements of the present invention include the compositions used with
 the embryo. For example, preferred compositions are adhesives, such as
 synthetic or biological adhesives. Biological adhesives have been used as
 hemostasis agents and for wound approximation. In surgical practice,
 biological adhesives are quite useful because they are easily prepared,
 they control bleeding quickly and effectively, they aid wound healing, and
 they leave little or no scarring. For these reasons, they have been used
 intensely in the past several years as replacements for sutures.
 Biological adhesives are preferable because they promote healing, and
 because they are readily absorbed by the body after use. Although recent
 FDA rulings allow such agents to be obtained from homologous sources,
 autologous materials should obviously be used if at all possible for
 optimum tissue matching. Most preferably, the adhesives of the present
 invention are made from the patient's own tissues. Other types of
 adhesives that are known to those skilled in the art are also contemplated
 in the present invention.
 One feature of the adhesives contemplated by the present invention is
 biocompatibility. The present invention contemplates both natural and
 synthetic adhesives that are biocompatible with either the embryo or the
 host, or both. Of the many such possible adhesives or glues that are
 contemplated by the present invention, the most preferred ones are based
 on collagen, albumin, platelets and fibrin. The platelet and albumin
 products have greater wound healing ability, but their bonds are
 inherently weaker than the bonds of fibrinogen products. Collagen, a
 connective tissue protein, can be obtained from both living and dead
 donors. Albumin, a highly nutritive blood component, is easy to collect
 from a living donor, but hard to separate on an autologous basis.
 Conversely, platelets and fibrin, which are parts of the normal clotting
 process, are preferable in this application because they can be collected
 and separated readily from a simple blood donation.
 Preferred methods for making these adhesives depends on the type of
 adhesive. For example, a platelet adhesive method begins with separating
 the platelets and plasma from the blood sample, as described in
 McCullough, Jeffrey, Transfusion Medicine, McGraw-Hill, 1998. The
 platelets are then processed into a gel form as known in the art for use
 as an adhesive in the present invention. The American Association of Blood
 Banks has regulations for making platelet gels and companies such as
 Medtronics commercially produce them. A preferred method for preparing
 autologous fibrin adhesives is fibrinogen cryoprecipitation. This process
 comprises freezing the plasma, followed by thawing to not greater than
 4.degree. C. Upon centrifugation, the white precipitate is then ready for
 use as a fibrin adhesive. An additional method step includes isolation of
 the autologous thrombin from the blood sample. Bovine thrombin can be used
 if the recipient has no previous allergic history to bovine proteins.
 A beneficial feature of biological adhesives is that the adhesive may be
 activated simply by the addition of thrombin. Furthermore, the rate of set
 of the adhesive is determined by the thrombin concentration. The clinical
 benefit is that these adhesives can be started when desired, manipulated
 into the desired shape, put in place and set, with all rates easily
 controlled. Commercially available fibrin adhesive kits are provided with
 two vials of thrombin, with the 10 IU vial used for slow sets over several
 minutes, and the 100 or 200 IU vial used for fast sets over several
 seconds. Synthetic adhesives are activated by other materials to provide
 the same level of control.
 Another beneficial feature of adhesives is that many are absorbed into the
 body, and this rate of absorption can be controlled by the addition of
 other agents. For biological adhesives, such agents include the addition
 of aprotinin in the composition. The clinical benefit is that the
 adhesives perform their tasks, and then disappear without additional
 action and without scarring. Another beneficial feature of adhesives is
 that various surface treatments diminish their adherence to such surfaces.
 This technique is clinically useful in preventing undesired attachments to
 instruments, gloves, surrounding tissues, and allows for manipulation of
 the adhesive compositions containing the embryos. One such surface
 treatment that can be used with biological adhesives is a concentrated
 saline solution.
 An advantage of platelet adhesives is that platelet adhesives are quite
 nutritious media, and cause no irritation or inflammation at the point of
 contact. Platelet adhesives are predictable in terms of bond strength, but
 their strength is relatively weak. Their strength is determined by the
 same .alpha. and .beta. integrin mechanism that determines the adhesive
 properties of the uterine wall relative to the embryo as described by
 Tabibzadeh, A., "Patterns of Expression of Integrin Molecules in Human
 Endometrium Throughout the Menstrual Cycle", Human Reproduction 7(6): 876,
 1992.
 Platelet adhesive compositions are contemplated by the present invention in
 part because platelet adhesives are well-suited for direct contact with an
 embryo due to their high nutritive value and inert bonding. Their
 relatively low strength of bonding is not much of a hindrance because
 small, light embryos require little adhesion.
 The present invention also comprises compositions comprising fibrin
 adhesives. Fibrin adhesives have comparatively little nutritive value, and
 are slightly irritating to tissues. An advantage of fibrin adhesives is
 their strength, which is greater than that of platelet adhesives. A
 disadvantage of fibrin adhesives is that the strength of adhesion is
 unpredictable. An explanation for the unpredictability is that the
 strength of the adhesive depends upon the square of the fibrinogen
 concentration. When the fibrinogen is obtained by a cryoprecipitate, the
 resulting composition has variable amounts of Factor VIII, von Willebrands
 factor, and other components at lesser concentrations. The present
 invention contemplates use of fibrin adhesive compositions comprising
 these factors and other proteins that are found in the cryoprecipitate.
 Another embodiment of the present invention includes the use of any
 biocompatible material in place of the fibrin substrate. The fibrin
 substrate can be replaced by an artificial substrate consisting of any
 biocompatible material, such as carbon blocks or fibers. Another option is
 to include a fibrin adhesive with a fiber matrix. The desired effect is a
 substrate that is easier to assemble and handle than a fibrin block alone.
 The low nutritive value and high strength of fibrin adhesives make them
 well suited for use as substrates and contact with the uterine wall, which
 has its own nutrition source. Though it might be thought that irritation
 due to the fibrin adhesive makes it necessary to avoid contact with the
 embryo, recent work suggests that some irritation actually improves
 implantation. See Fioretti, et al, "Fusion Gene Products Encoding Avian
 Alpha Subunit Inhibin protein, or an Immunogenic Fragment Thereof, and a
 Carrier Protein", U.S. Pat. No. 5,747,659; 1998. Thus, contact by the
 fibrin adhesive with the uterine wall is beneficial. Another embodiment of
 the present invention is to mix the platelet and fibrin adhesives, thereby
 providing a range of strengths, nutritive values, and site irritations. A
 preferred method of the present invention comprises having the embryo in
 close contact with the more nutritious platelet or albumin products, while
 the fibrin products are used to provide greater strength over longer
 distances.
 The adhesive compositions can be applied to the embryo by using any method
 that allows for control of the adhesive with little manipulation of the
 embryo. A preferred method is to directly apply the adhesive composition
 to embryos that are stabilized in an embryo preparation plate. Another
 preferred method is to first pour the adhesive composition on a surface
 and allow the adhesive composition to begin setting up. Second, remove a
 plug of the adhesive composition that has begun setting and place that
 plug over the embryo.
 Additional adhesive compositions can be added to the embryo as needed to
 provide support, adhesion or nutrition. For example, after placing a
 platelet adhesive composition plug over the embryo, a fibrin adhesive
 composition is poured over the plug/embryo structure so that the fibrin
 adhesive composition is the outer surface that touches the uterine wall.
 A preferred method of encasing embryos for implantation comprises the
 following steps. An adhesive composition is made and allowed to begin
 setting. The embryo or embryos are then placed in an embryo preparation
 plate.
 The plate has been prepared so that the adhesives will not stick to the
 plate. For example, preparation comprises rinsing the plate with a saline
 solution for easy release of the adhesive. Next, the embryo is placed
 within a depression in the plate, preferably using micromanipulators,
 though any known methods of embryo handling equipment are contemplated by
 the present invention. If the embryo has undergone mechanical assisted
 hatching, it is placed so that the compromised part of the zona pellucida
 is facing the bottom of the depression. No particular orientation of the
 embryo is necessary for the methods of the present invention and any
 particular required orientation, for example, required in order to
 facillitate implantation, is contemplated by the present invention.
 The adhesive plug is added to the embryo. For example, using an adhesive
 probe such as the one described in Example II, the cylindrical probe
 containing the adhesive is then centered over the isolated embryo. The
 syringe is pressed, thus expelling the adhesive plug. This plug, which is
 still quite fluid, flows over the embryo and begins to adhere to the
 embryo wall. The plug is sufficiently set so that it cannot flow into the
 small space between the embryo and the hemispherical depression. The net
 result is an embryo with only a selected area covered by a desired amount
 of adhesive.
 In another embodiment, two adhesives may be used. The first adhesive is
 applied in the manner just described for plug attachment to the embryo.
 Any of the adhesives, natural or synthetic can be used, though a preferred
 composition comprises a platelet adhesive. A second adhesive is then
 applied. Any of the adhesives, natural or synthetic can be used, though a
 preferred composition comprises a fibrin adhesive. FIGS. 1 and 3 show the
 resulting embryo encasement using two adhesives.
 This method, comprising addition of two adhesives, comprises a first step
 that uses a probe that deposits an adhesive composition, including but not
 limited to, a platelet gel layer, on the embryo, and removal of the probe.
 Next, a larger diameter probe, preferably mounted concentric to the
 embryo, is used to apply the second adhesive, including but not limited
 to, a fibrin adhesive. This second probe may also contact a raised ring
 beyond the boundary of the platelet gel, thus forming a protrusion in the
 fibrin glue directly over the embryo.
 There are many immediate benefits to this two adhesive method, beginning
 with the improved surface contact provided by two different adhesives.
 Specifically in this preferred embodiment, the adhesive in direct contact
 with the embryo is a platelet gel adhesive and the second adhesive is a
 fibrin adhesive. In addition, the fibrin glue surface beyond the embryo is
 free to contact the uterine wall.
 The unique geometry illustrated in FIG. 3, yields several additional
 benefits. One such benefit is that the fibrin adhesive layer provides a
 durable substrate for the weaker platelet gel layer, thus maintaining the
 desired geometry during transfer of the embryo to the uterine site. In
 turn, a key feature of this geometry is the protrusion illustrated in the
 side view of FIG. 3. This protrusion, which is below the elevated fibrin
 adhesive layer in contact with the uterine wall, forces the embryo and
 uterine surfaces into very tight contact and improves the chances of
 implantation. This tension is maintained over time because the durability
 of the substrate prevents the embryo from recessing excessively into the
 glue layers.
 Another aspect of the present invention is that after the embryo is
 attached to the adhesive composition or compositions, the embryo at this
 point is contained within the steel cylinder of the probe. Upon lifting
 the imbedded probe from the surface, the embryo is usually slightly
 recessed, thereby protecting the embryo from accidental side contact and
 subsequent disruption.
 Another element of the present invention is an applicator to place the
 adhesively imbedded embryos within the uterus and attach them to the wall.
 Features of this device include a means of holding the embryos embedded in
 adhesive, a means of guiding the device to the desired location and a
 means of uterine wall attachment.
 Of these components, the first is the holding device. The delicate embryo
 must be protected during transfer, but released when necessary. This can
 be achieved by including retractable metal retainer pins, as indicated in
 FIG. 1. These pins, which are sprayed with saline solution for easy
 release, allow the adhesive layer to be lifted easily from the plate,
 along with the embedded embryos.
 With the pins in place in the applicator, the embryos can then be held at a
 safe distance from a retractable metal plate, which provides protection
 from contact with the vaginal or cervical walls during transfer.
 During transfer, the assembled applicator and embryo/adhesive load can be
 guided either by ultrasound or by direct visualization with a
 hysteroscope. Upon reaching the desired location within the uterus, the
 embryo and adhesive mixture are then attached with an additional
 application of an adhesive such as a fibrin adhesive. The adhesive and
 embryo patch is held closely against the uterine wall by either spring or
 jack force during this gluing process, thereby preventing additional
 fibrin glue from seeping into the desired attachment point. The applicator
 may also incorporate separate activation wires.
 Several other embodiments are possible for this method and device. The
 applicator can incorporate separate guide wires to steer the device to the
 desired target with minimum trauma to the uterine walls. These guide wires
 must be oriented to maintain their desired relative positions while they
 reverse direction upon passing through the vagina, into the cervix, and
 into the uterine cavity.
 The rate of absorption of the adhesive can be slowed by the addition of
 aprotinin to the adhesive compositions, or increased by the addition of
 various clot-dissolving enzymes to the adhesive compositions, depending on
 the conditions required for a given patient.
 The adhesive compositions could be intermixed, thereby simplifying the
 process. For example, in surgical uses, mixed platelet and fibrin
 adhesives already show some promise for providing both enhanced healing
 and strength. The adhesives can also incorporate additional nutritive
 agents and/or antibiotics.
 The present invention comprises methods of gluing the embryos to the
 uterine wall. The net result is that the embryos are in direct contact
 with a selected point in the uterus for a long period of time. By using
 biologically compatible materials, rejection is avoided during this
 process.
 It is generally accepted that reduction or elimination of the reduced
 contact associated with "floating" of the embryo such as are used during
 prior implantation procedures should be helpful.
 Another embodiment of the present invention comprises the use of a transfer
 device with an applicator. Having prepared the embryo with adhesives, the
 next step is to transfer this embryo to the patient's uterus. In a
 preferred embodiment, the transfer device consists of two components: a
 loader to first gain access to the uterus, and next, an applicator to glue
 the embryo in place.
 The first step of this transfer process is to gain access to the uterus in
 a safe, sterile and efficient manner. Current practices use a combination
 of germicidal agents, specula and guide channels. This approach
 occasionally results in infections and cannot manipulate embryos treated
 as described herein. A method of the present invention comprises use of
 germicidal agents and specula, and a unique guide channel through the
 cervix. In order to comply with anatomical demands and to prevent
 infections during implantation, the transport loader of FIG. 4 can be
 used.
 As shown in FIG. 4, this device comprises an outer barrel that is flared at
 one end to prevent over insertion. Inside this barrel is a matching
 plunger of slightly smaller diameter. A thin film, such as a plastic film,
 rolled within the plunger, joins the far, or distal ends of the barrel and
 plunger. The far or distal end of the plunger has three matching leaves,
 meeting at a dull point.
 The first step in using this device is to bend the flexible barrel and
 plunger into the desired curvature, and then insert the barrel as far as
 possible into the cervix. The plunger is then pressed, which further
 penetrates the cervix. Unlike conventional probes, however, the enclosed
 film unrolls during this process. Because this film, instead of the
 plunger itself, contacts the cervix, there is no entrainment of
 contaminants. This continuous dispensing of the film also prevents the
 delicate cervical tissues from being drawn along with the plunger, thereby
 increasing patient comfort. The pointed end of the plunger also improves
 patient comfort by gradual opening of the channel. Upon full insertion
 into the uterus these leaves flare outward, providing a secure anchor
 effect and a tapered entry and exit path, which aids in the insertion and
 removal of the applicator described below.
 Additional embodiments of this device include germicidal and/or anesthetic
 agents on the film, thus resulting in smooth, uniform application. With
 such modifications, this device could thus be used in a wide variety of
 gynecological procedures, as well as various urologic and respiratory
 applications.
 Having gained access to the uterus through the hollow plunger, the next
 step is to attach the embryos in place on the uterine wall. This is the
 task of the second component of the transfer instrument, the applicator.
 Like the above loader, this component requires anatomical considerations. A
 preferred instrument comprises a small, flexible device, capable of
 negotiating the narrow cervical canal while adapting to the angle between
 the vagina and uterus. The simplest such device is a thin plastic tube,
 like those currently used in IVF practice. In current practice, however,
 the suspended embryos are simply squirted into the uterus through this
 tube, but this technique will not work with embryos in adhesives.
 Furthermore, the current technique provides no control of the implantation
 site, whereas the present invention allows for embryo attachment at a
 selected location.
 One alternative is to attach the embryo to the end of the plastic tube, and
 then insert the tube all the way through the uterine cavity to the
 opposing wall. The adhesive would then bond to the wall, and the
 instrument would be withdrawn. There are essential considerations that
 must be taken into account when using this method, such as determining the
 proper insertion depth. If the probe is only millimeters or even less away
 from the wall, the glued embryo will not attach properly, leading to
 implantation failure. Conversely, if the probe is inserted too deeply, the
 embryo could be forced into the glue or possibly even crushed. Even worse,
 slightly deeper insertion could result in uterine perforation, a very
 dangerous complication. With proper control, such as use of viewing
 instruments and/or tactile control, this method of implantation can be
 utilized.
 The central mid-section of the uterus is the desired implantation site, and
 an applicator that can provide embryos for attachment at this site is
 preferred. To reach these walls, the implantation unit must therefore be
 capable of gluing the embryo at right angles, but action at right angles
 is mechanically difficult.
 In an embodiment of the device of the present invention, mounting the glue
 application barrel at right angles to the insertion probe resolves the
 action angle problem. The barrel can be bent or miter cut to form a
 90.degree. angle. Although the hydraulic drive on the plunger works
 through any angle, there is still the potential problem that the barrel
 may not be aligned normal to the wall plane, thereby not properly placing
 the embryo. There is also the potential problem that the curvature of the
 flexible probe may leave the barrel too far away from the uterine surface
 for effective placement.
 The application instrument can be modified to allow embryo placement at the
 top as well as the bottom of the inserted unit. The advantage of this
 approach is that the embryos can be placed on opposite sides of the
 uterus, which is the ideal placenta location for twins in case both
 embryos implant.
 A device of the present invention mounts a pair of balloons on opposite
 sides of the insertion probe, as illustrated in FIG. 5. Using the
 technology employed for balloon angioplasty, the opposed balloon pair
 thereby fills the intrauterine space. The inflatable balloon may include
 reinforcing ribs so that all of the balloon material can be safely removed
 even if the balloon ruptures. This fixed alignment of the balloons aligns
 the probe directly at the wall. To ensure proper wall contact, fixed bands
 on the side of the balloons, near the barrel exit, force the expansion in
 the opposite direction. Complete expansion forces the barrel directly, but
 gently, against the uterine wall. Upon complete expansion, plastic strips
 glued to the bottom of the balloons on opposite sides of the barrel
 maintain the barrel at the proper location throughout the attachment
 process. In addition, these strips act as skids, allowing the unit to be
 repositioned by pulling or pushing the guide wire even with the balloons
 inflated.
 There is a sheath around the entire guide wire, to form a conduit from
 outside the body all the way to the applicator tip. This conduit provides
 a means of loading prepared barrels after the applicator is in place,
 thereby avoiding potential loss of the embryo during the insertion and
 placement of the applicator. In addition, this device also provides a
 means to attach more than one embryo without removing and replacing the
 entire applicator.
 In summary, the methods of the present invention comprise a first step of
 preparing the embryos with adhesives, and then containing each embryo in a
 separate barrel, tube and syringe assembly. Simultaneous to this
 laboratory work, the patient is to be prepared in the clinic. As
 described, the first clinical step is to insert the loader into the
 patient. Next the transfer applicator is inserted and guided into place
 under ultrasound imaging. Fiber optic viewing can also be used for correct
 placement. Once the applicator is in place, the balloons are inflated.
 Using the current patient/embryo identification matching procedures, the
 first prepared embryo is handed from the laboratory technician to the
 attending physician. The physician passes the mounted embryo through the
 conduit along the guide wire until the barrel assembly reaches the far tip
 of the instrument. The saline syringe will then be activated, thus
 displacing the plunger and forcing the glued embryo into contact with the
 uterine wall. This configuration is maintained while the glue bonds to the
 wall, for approximately 2-20 minutes, preferably about 2-10 minutes and
 most preferably, about 3 to 5 minutes. The saline syringe is then pulled
 back to retract the plunger and release the embryo. The guide wire is
 withdrawn about 1 cm, drawing the balloon assembly along on its skids. The
 entire applicator passes over the attached embryo without risk of
 dislodging it.
 This attachment method is then repeated for the next embryo or embryos.
 After the last embryo is in place, the balloons are deflated, and the
 applicator is removed through the loader. The loader is then removed from
 the cervix, completing the transfer procedure.
 It is contemplated by the present invention that embryos prepared with the
 adhesive compositions of the present invention can be implanted into the
 patient by any means known to those skilled in the art and the present
 invention is not limited by the devices or methods for implantation taught
 herein. Additionally, the present invention contemplates that the methods
 and devices for implanting embryos that are taught herein can be used for
 other procedures involving humans or animals wherein a controlled
 manipulation within an internal space is needed. For example, such
 controlled manipulations may be needed in urinary, gastric or other
 gynecological procedures.
 This invention is further illustrated by the following examples, which are
 not to be construed in any way as imposing limitations upon the scope
 thereof. On the contrary, it is to be clearly understood that resort may
 be had to various other embodiments, modifications, and equivalents
 thereof which, after reading the description herein, may suggest
 themselves to those skilled in the art without departing from the spirit
 of the present invention and/or the scope of the appended claims.
 EXAMPLES
 Example 1
 A Method of Adhesive Attachment
 An embodiment of a preferred method is described here and the device used
 was as shown in FIG. 1. A test cell mass, that resembles an embryo in size
 and cellular fragility, was used in place of an actual embryo. With the
 test cell mass in position in the hemispheric depressions of the embryo
 preparation plate, a thin layer of platelet or albumin adhesive was
 applied. Because of the viscoelastic behavior of these materials, only the
 top half of the test cell mass was coated. This arrangement complemented
 the half-submerged geometry of normally implanted embryos. This coating
 procedure would leave any AH sectors uncovered, given the orientation
 described above.
 Next, a thin layer of fibrin glue was applied, consisting of a mixture of
 fibrinogen and thrombin. This mixture was dispensed by conventional dual
 cylinder applicators. For rapid clot formation, approximately 200 units of
 thrombin were used for each cc of applied fibrinogen.
 A metallic sleeve was then placed around the half-coated embryo,
 penetrating down to the base of the incubation plate. For easy release
 from the fibrin bonds, this sleeve was sprayed with saline solution.
 The sleeve was either small and circular in cross section to remove a
 single test cell mass, or large and extended to remove an entire set of
 test cell masses. In either case, the net result was that the test cell
 masses were half-submerged in an adhesive layer. This layer was readily
 lifted from the incubation plate, with the test cell masses exposed on one
 common side, which was again oriented downward for AH.
 Example 2
 A Method of Encasing an Embryo in Adhesives
 The method was to form a thin layer of adhesive and then this layer was
 attached to one side of the test cell mass. To accomplish this, first, the
 adhesive components were mixed. Next, a thin layer of glue was spread on a
 flat Teflon surface, which was previously rinsed with saline solution for
 easy glue release. The glue was then allowed to begin to harden, as
 evidenced by the formation of striations of varying thickness across the
 surface.
 A probe was used to extract a specified amount of the glue in a specified
 shape. This probe was constructed of a 2 mm length of stainless steel cut
 tube from a hypodermic syringe. The remaining part of the syringe was then
 forced through a 1.5 mm thick layer of rubber, thus forming a sealed
 plunger. This plunger was then inserted into the probe, where it was
 retained by a crimp at each end. The far end of the probe was attached to
 a 15 cm length of flexible plastic tubing, the opposite end of which was
 connected to a syringe filled with saline solution. Pressing and pulling
 the syringe plunger thus caused the rubber plunger inside the probe to
 move the entire length of the barrel in the corresponding direction. The
 system was then cycled repeatedly at high syringe pressure so that the
 rubber plunger was eroded sufficiently to protrude 0.75 mm beyond the tube
 at maximum extension.
 When no further striations develop in the adhesive, the assembled probe was
 inserted through the thickest part of a striation, progressing slightly
 beneath the plastic surface. The remaining adhesive, which had not yet
 reached the point of tack, was then rinsed from the plate. Without the
 surrounding adhesive, the probe can then be lifted cleanly from the plate
 surface. When operated with pre-mounted jigs, this procedure took a few
 seconds to complete.
 The net result was a small, well-defined plug of adhesive. This plug was
 allowed to remain in the probe barrel for a few minutes and approach tack.
 The next step was to apply the resulting cylindrical plug layer of glue
 onto the test cell mass. Use of this type of adhesive plug is shown in
 FIG. 2.
 It should be understood, of course, that the foregoing relates only to
 preferred embodiments of the present invention and that numerous
 modifications or alterations may be made therein without departing from
 the spirit and the scope of the invention as set forth in the appended
 claims.