WO2001017443A1

WO2001017443A1 - A method and device for artificial insemination

Info

Publication number: WO2001017443A1
Application number: PCT/IL2000/000531
Authority: WO
Inventors: Tamar Tennenbaum
Original assignee: Tamar Tennenbaum
Priority date: 1999-09-09
Filing date: 2000-09-05
Publication date: 2001-03-15
Also published as: IL131826A0; AU6862500A

Abstract

The invention provides a method for sperm preparation and artificial insemination comprising placing a sperm sample in a syringe-like container (4) having a narrow first opening (6) with a removable seal (8), and a wider second opening (10), introducing a plunger-like device (12) into the second opening, wherein the device (12) is provided with a filter element (14) along a bottom surface thereof, the filter leading to a chamber (16) in the plunger (12), and wherein the device is sized for friction-fit engagement with inner walls (18) of the container (4), whereby a portion of smaller components of the sample, including seminal fluid, proteins, prostaglandins and bacteria are forced through the filter (14), into the chamber (16), leaving motile sperm cells and a portion of seminal fluid in the container, at least partially removing the plunger-like device (12) and discarding filtrate collected therein, introducing pure medium into the container to wash sperm cells and dilute seminal fluid remaining therein, depressing the plunger-like device into the container (4) to force at least a portion of the medium and remaining inpurities into chamber thereof, thereby leaving washed motile sperm cells and a portion of the medium in the container, at least partially removing the plunger-like device and discarding filtrate collected therein and utilizing washed motile sperm cells remaining in the container for artificial insemination.

Description

A METHOD AND DEVICE FOR ARTIFICIAL INSEMINATION

The present invention relates to a method for artificial insemination More particularly, the present invention relates to a metnod and device for sperm preparation and artificial insemination

Approximately 15% of the couples who want to have a child are infertile Infertility is defined as the inability to achieve pregnancy within one year of sexual intercourse without contraceptives

From data collected during the past twenty years, it was found that in 45% of the infertile couples' infertility is due to a male factor In cases where infertility was due to a female factor, it could be classified into impairments in ovulation, a mechanical factor, such as occlusion of the oviducts, endometnosis a cervix factor or unexplained infertility In males infertility factors can be classified into semen quality, including number, motihty and structure of sperm cells anti-sperm antibodies, infections, structural defects in the male vaπcocele, problems in ejaculation and unexplained infertility

The most common primary treatment to sperm cells that is suggested to the couple (depending on the type of problem) is sperm preparation and insemination of the woman (with spontaneous ovulation or following hormone treatment) The term 'sperm preparation' is a general name for several processes that prepare the semen for artificial insemination In this process, the sperm cells are separated from the seminal plasma Sperm preparation is also used for separating and extracting the motile and normal sperm cells from defective and/or immotile cells and from accompanying cells such as bacteria or white blood cells

Sperm preparation is performed either in a laboratory adjacent to the physician's office or, in many cases, in an external sperm laboratory

Artificial insemination of normal sperm to a fertile woman will lead to pregnancy in a period of between 6 and 12 months Usually, the woman is inseminated twice to three times near the time of ovulation, such that when the ovum reaches the fertilization site sperm cells will be present at the site

The physician can perform insemination in three places in the female genital tract A Intravaginal Insemination - The semen are injected into the vagina near the cervix This method can also be performed by the couole themselves B. into the Cervix - The semen are injected into the cervix.

C. Intrauterine Insemination OUT) - Insemination directly into the uterine cavity in order to introduce the sperm cells as ciose as possible to the fertilization site. Common techniques for sperm preparation are known and are described in various publications (See, e.g. Glezerman, M. (1993). Artificial insemination. In: Infertility Male and Female. Insler, V., Lunenfeid, B. (Eds.) p. 643; Utto, 1992; Allen, N.C., Herbert, CM., Maxson, W.S., Rogers, B.J., Diamond, M.P. and Wentz, A.C. (1998). Intrauterine insemination. In: Modem Trends in Infertility and Conception Control. Vol. 4, Wailach, E.E., Komps, R.D. (Eds.) p. 487; Allen, N.C., Herbert, CM., Maxson, W.S., Rogers, B.J., Diamon, M.P. and Wentz, A.C. (1985). Intrauterine Insemination: A Critical Review. Fertil. Steril. 44:56; Mortimer, D. (1990). Semen Analysis and Sperm Washing Techniques. In: Controls of Sperm Motility: Biological and Clinical Aspects. Gagnon, C. (Ed.); WHO - World Health Organization (1992). Laboratory Manual for the Examination of Human Semen and Sperm Cervial Mucus Interaction.) include: A. Dilution and Washing: Sperm dilution is performed by the addition of a large volume of synthetic seminal fluid, (such as Ham F-10) mixing and subsequent washing by centrifugation. The relatively heavy sperm cells accumulate on the bottom of the test tube whereas the seminal fluids remain in the supernatant. Removal of the supernatant and exchanging it with synthetic seminal fluid and repetition of the washing process cleans the sperm from the seminal fluid. However, this method, while used by most gynecologists in preparing sperm cells for intrauterine insemination, does not separate the normal sperm cells from the defective sperm cells. B Swim-Up Technique: After ejaculation and introduction of the semen in the vagina by the male, the sperm cells exhibit strong movements as they travel from the vagina to the cervical mucus and into the uterus and thus, they are separated from the seminal fluid, as well as from the immotile sperm cells. The swim-up technique imitates this process in vitro. In this method the sperm cells are able to migrate into the supernatant found above the sperm cell suspension. After incubation, the supernatant containing the motile sperm cells is used for intra-uterus insemination (I.U.I.) or In-vitro fertilization (I.V.F.) C. Washing Using a Density Gradient (for example Ficoll): This method is based on centrifugation of the sperm through a fluid with an increasing density gradient, in order to separate sub-populations of the sperm cells in the semen. Centrifugation with this method results in the separation of motile sperm cells having a normal condensed head.

These cells are separated from non-condensed sperm cells, bacteria and white blood cells that are found in the semen.

D. Preparation of Sperm by Passage Through a Column (known by the commercial name of SpermPrep or SpermFertil): This method enables separation of motile sperm cells from those that are immotile and from most of the white blood cells. In this method, the motile sperm cells with the condensed head, leave the column relatively fast whereas immotile sperm cells and white blood cells are adsorbed onto the column.

With this state of the art in mind, there is now provided according to the present invention, a method for sperm preparation and artificial insemination comprising: a) placing a sperm sample in a syringe-like container having a narrow first opening with a removable seal, and a wider second opening; b) introducing a plunger-like device into said second opening, wherein said device is provided with a filter element along a bottom surface thereof, said filter leading to a chamber in said plunger, and wherein said device is sized for friction-fit engagement with the inner walls of said container, whereby a portion of the smaller components of said sample, including seminal fluid, proteins, prostaglandins and bacteria are forced through said filter, into said chamber, leaving motile sperm cells and a portion of seminal fluid in said container; c) at least partially removing said plunger-like device and discarding the filtrate collected therein; d) introducing pure medium into said container to wash the sperm cells and dilute the seminal fluid remaining therein; e) depressing said plunger-like device into said container to force at least a portion of said medium and remaining impurities into the chamber thereof, thereby leaving washed motile sperm cells and a portion of said medium in said container; f) at least partially removing said plunger-like device and discarding the filtrate collected therein; and g) utilizing the washed motile sperm cells remaining in said container for artificial insemination.

Preferably, said filter is in the range of between 1.3 and 3.5 microns and especially preferred, is a filter within the range of between 1.8 and 3 microns.

In preferred embodiments of the present invention said seal is removed from said first opening after step (f), and step (g) is carried out by inserting a piston-like element into said container to force the washed motile sperm cells through said first opening for effecting the artificial insemination step.

In another aspect of the present invention, there is provided a device for carrying out the above method comprising: a) a syringe-like container having a narrow first opening with a removable seal and a wider second opening; b) a tubular plunger-like device provided with a filter element along the bottom surface thereof said filter leading to a chamber in said device, said device being sized for friction fit engagement with inner walls of said container. The arrangement being such that, upon introduction of said device into said second opening and depression thereof into said container, a portion of the smaller components of a sperm sample previously placed in said container, are forced through said filter into said chamber, leaving motile sperm cells and a portion of seminal fluid in said container.

In preferred embodiments of this aspect of the present invention there is provided a device as defined above in combination with a piston-like element sized for friction fit engagement with the inner walls of said chamber and adapted to effect the sealing of said filter when depressed into said chamber to force washed motile sperm cells through said first opening after the removal of said seal therefrom.

The invention will now be described in connection with certain preferred embodiments with reference to the following illustrative figures and in the following examples, so that it may be more fully understood. With specific reference now to the figures in detail, it is stressed that the particulars shown are by way of example and for purposes of illustrative discussion of the preferred embodiments of the present invention oniy and are presented in the cause of providing what is believed to be the most useful and readily understood description of the principles and conceptual aspects of the invention. In this regard, no attempt is made to show structural details of the invention in more detail than is necessary for a fundamental understanding of the invention, the description taken with the drawings making apparent to those skilled in the art how the several forms of the invention may be embodied in practice.

In the drawing:

Figure 1 is a cross-sectional view of a preferred device according to the present invention.

Referring to figure 1 , there is seen a device (2) according to the present invention, comprising a syringe-like container (4), having a narrow first opening (6) with a removable seal (8) and a wider second opening (10). Also provided, is a plunger-like device (12) provided with a filter element (14) along the bottom surface thereof said filter leading to a chamber (16) in said plunger, wherein said device is sized for friction fit engagement with the inner walls (18) of said container (4) and preferably provided with o - ring seals (20), for assuring a leak-proof friction fit. Also provided, is a piston-like element (22) sized for friction fit engagement with the inner walls of said chamber (16) which is used to seal said filter (14) and depress said plunger like device (12) thereby to force washed motile sperm cells through said first opening (6) after the removal of seal (8) therefrom, after the sperm cells in the container have been prepared and washed, as described hereinafter.

EXAMPLES

METHODS

The study group consisted of a random group of eight men with suspected infertility.

The single semen examination for each member of the study group was performed according to the WHO (1992) laboratory manual for semen analysis, after 4 days of sexual abstinence. The following sperm characteristics were examined: sperm count, percentage of motile spermatozoa, degree of motility (0-4) and percentage of normal forms. The semen samples were diluted at a ratio of 1 :1 in HamF-10 fluid. The diluted semen were divided into two equal parts. One part underwent sperm preparation using the washing technique of centrifugation for ten minutes at 1600 rpm; and the second part underwent sperm preparation using the device and method of the present invention. (Hereinafter referred to as the Washing By

Filtration Technique) (The semen was filtered twice and washed in HamF-10 using the device as described, with reference to the appended figure.)

Sperm count, motility and sperm cell structure were examined in each of these prepared semen samples. After examination, the semen were centhfuged again at 3000 rpm for 30 minutes and the supernatant was examined for the presence of seminal plasma components - citrate and fructose.

The Washing by Filtration Technigue: Washing of sperm cells was performed by: a) placing a sperm sample in a syringe-like container (4) having a narrow first opening (6) with a removable seal (8), and a wider second opening (10); b) introducing a plunger-like device (12) into said second opening, wherein said device is provided with a filter element (14) along a bottom surface thereof, said filter leading to a chamber (16) in said plunger, and wherein said device is sized for friction-fit engagement with the inner walls (18) of said container, whereby a portion of the smaller components of said sample, including seminal fluid, proteins, prostaglandins and bacteria are forced through said filter (14), into said chamber (16), leaving motile sperm cells and a portion of seminal fluid in said container; c) at least partially removing said plunger-like device (12) and discarding the filtrate collected therein; d) introducing pure HamF-10 medium into said container (4) to wash the sperm cells and dilute the seminal fluid remaining therein; e) depressing said plunger-like device (12) into said container to force at least a portion of said medium and remaining impurities into the chamber thereof, thereby leaving washed motile sperm cells and a portion of said medium in said container and f) at least partially removing said plunger-like device (12) and discarding the filtrate collected therein. In this procedure, the sperm sample was initially placed in said container, already diluted with HamF-10 medium.

The filter enabled passage and removal of the seminal fluids, various substances such as proteins or prostagiandins, bacteria and cell fragments, but did not enable the passage of sperm cells.

The Washing by Centrifugation Technigue: The semen were diluted at a ratio of 1 :1 and centhfuged at 1600 rpm for ten minutes. The supernatant was removed and substitute fluid HamF-10 was added to a volume of 1 ml. The washed semen underwent incubation at 37°C for an additional 10 minutes before counting. RESULTS

The aim of these experiments was to compare the sperm quality , after preparation of the semen using the technique of washing by centrifugation and after washing by filtration according to the present invention, to those of untreated semen.

1. Total sperm count :

No significant difference was found in the number of sperm cells after both treatments.

2. Motility:

It was found that motility following washing by filtration was not lower than the motility following centrifugation, and was even significantly improved compared to the sperm cell motility in the original semen (p<0.05). No significant difference was found in sperm cell motility before treatment and after centrifugation, as seen in Table 1.

TABLE 1

3. Morphology:

No difference was found in the percent of normai shaped sperm cells in the three above-mentioned groups. Furthermore, no significant difference between the sperm cells was found in neck and tail malformation.

However, a significant increase in head malformations was found in sperm cells that underwent centrifugation, compared to those that underwent filtration. No significant differences were found in head malformations between the sperm cells that underwent filtration and those in the natural semen.

The device for washing sperm cells, presented here, was developed in order to replace the sperm washing technique by centrifugation. This device and the present method will enable exchanging the seminal plasma by a synthetic medium simply, easily, rapidly, with no need for heavy equipment (such as a centrifuge), elegantly and in a sterile fashion, e.g., the washed seminal plasma may optionally be directly placed in closed vessels for disposal. The kit is composed mainly of fluids and vessels that are commonly used in the health systems.

From the results, it can be seen that sperm preparation, using the filtration washing technique of the present invention gives good results. The quality of the sperm cells obtained after washing by filtration, is at least as good as the quality obtained after centrifugation.

The device and method of the present invention can also be compared to the technique of passing the semen through columns. However, these two techniques differ in essence. The washing device and method of the present invention exchanges the natural seminal fluid with a synthetic fluid. This does not separate between the sperm cell population, i.e. it does not separate a good and motile sperm cell from other components in the semen. In contradistinction, the column is intended for separating motile and condensed sperm cells from immotile sperm cells and from white blood cells in the semen with the motile cells passing through the columns and then being collected for use.

However, the device presented herein, is easy to use, quicker (a few minutes as compared to approximately 30 minutes); and does not require an authorized and proficient laboratory team. The column can only be used in the laboratory and most physicians currently do not use this technique. The suggested method has numerous advantages. The physician can perform the preparation in the presence of the couple and can inseminate the woman with the husband's sperm ceils without removal thereof from the container in which the washing and purification has taken place, by simply removing the seal from the narrow first opening of the device and inserting a piston-like plunger for expelling the washed motile sperm cells remaining in said container directly into the woman to be inseminated.

A. The technique is relatively rapid: Performance of the procedure using the device of the present invention , will last a few minutes, compared with the existing methods which last from half an hour to an hour.

B. The physician can give the couple the security of knowing that the sperm is their own: The semen undergoes preparation in their presence, as opposed to other preparation techniques where the procedure is not performed in the presence of the couple (this also has important implications according to Jewish religious law).

C. The physician saves the couple trips to the laboratory and back: The existing procedure today is that the physician sends the couple to a laboratory. The couple leave the physician's clinic and drive to the laboratory (which is not always nearby). The man gives semen in the public toilets in the laboratory (a process that is not at all pleasant). The preparation is performed in the laboratory and, after waiting for approximately one hour, the prepared sperm is returned to the couple. The couple go back to the physician for insemination of the prepared sperm. Sometimes they have to wait again before being admitted to the physician. This waiting period may be significant for the sperm cell quality and motility. It is recommended that the insemination be repeated two to three times at the time of ovulation. The suggested kit removes the need for these trips, as well as the unpleasantness they involve and enables the gynecologist to give his client better service.

D. The physician does not need heavy equipment, such as a centrifuge.

E. This technique increased the physician's reliability.

F. The device can save costs.

In U.S. patents 5,028,526 and 5,185,246, both to Alice Deutsch, there is described a method for semen analysis involving the separation of seminal plasma from semen by means of a membrane, however neither of said patents teaches nor suggests a method and device for artificial insemination as described and claimed herein. it will be evident to those skilled in the art that the invention is not limited to the details of the foregoing illustrative embodiments and that the present invention may be embodied in other specific forms without departing from the spirit or essential attributes thereof. The present embodiments are therefore to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein.

Claims

WHAT IS CLAIMED IS:

1. A method for sperm preparation and artificial insemination comprising: a) placing a sperm sample in a syringe-like container having a narrow first opening with a removable seal, and a wider second opening; b) introducing a plunger-like device into said second opening, wherein said device is provided with a filter element along a bottom surface thereof, said filter leading to a chamber in said plunger, and wherein said device is sized for friction-fit engagement with the inner walls of said container, whereby a portion of the smaller components of said sample, including seminal fluid, proteins, prostaglandins and bacteria are forced through said filter, into said chamber, leaving motile sperm cells and a portion of seminal fluid in said container; c) at least partially removing said plunger-like device and discarding the filtrate collected therein; d) introducing pure medium into said container to wash the sperm cells and dilute the seminal fluid remaining therein; e) depressing said plunger-like device into said container to force at least a portion of said medium and remaining impurities into the chamber thereof, thereby leaving washed motile sperm cells and a portion of said medium in said container; f) at least partially removing said plunger-like device and discarding the filtrate collected therein; and g) utilizing the washed motile sperm cells remaining in said container for artificial insemination.

2. A method according to claim 1 , wherein steps (d), (e) and (f) are repeated.

3. A method according to claim 1 , wherein said filter is in the range of between 1.8 and 3 microns.

4. A method according to claim 1 , wherein said seal is removed from said first opening after step (f) and step (g) is carried out by inserting a plunger into said container to force the washed motile sperm cells through said first opening;

5. A method according to claim 1 wherein said sperm sample is diluted in medium, in said container, in step (a).

6. A device for carrying out the method of claim 1 comprising: a) a syringe-like container having a narrow first opening with a removable seal and a wider second opening; b) a tubular plunger-like device provided with a filter element along the bottom surface thereof said filter leading to a chamber in said device, said device being sized for friction fit engagement with inner walls of said container, the arrangement being such that upon introduction of said device into said second opening and depression thereof into said container, a portion of the smaller components of a sperm sample previously placed in said container, are forced through said filter into said chamber, leaving motile sperm cells and a portion of seminal fluid in said container.

7. A device according to claim 6 in combination with a piston-like element sized for friction fit engagement with the inner walls of said chamber and adapted to effect the sealing of said filter when depressed into said chamber to force washed motile sperm cells through said first opening after the removal of said seal therefrom.