RU2762489C1 - Method for isolating spermatozoa from aspiration and/or biopsy material from an appendage and/or testicle for use in in vitro fertilization and/or cryopreservation programs - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention relates to medicine, namely to reproductology, andrology and clinical embryology. A method for isolating spermatozoa from aspiration and/or biopsy material from an appendage and/or testicle for use in in vitro fertilization and/or cryopreservation programs involves separating spermatozoa from other cellular elements of the obtained material by at least two cycles of centrifugation with selection for each subsequent cycle of centrifugation of the supernatant obtained as a result of the previous cycle of centrifugation. Moreover, centrifugation is carried out with 200-350 g for 1-2 minutes. The cycles of centrifugation of the supernatant are carried out until there is no visible sediment.

EFFECT: invention provides for the production of a pure fraction of spermatozoa that can be used for fertilization of oocytes by ICSI and/or cryopreservation, from epididymis aspirate or testicular biopsy, including with their critically small amount.

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Description

The invention relates to medicine, namely to reproductive medicine, andrology and clinical embryology.

In the structure of the causes of infertility in couples, about half of the cases are associated with impaired fertility in men. To a serious factor of male infertility include azoospermia - a pathological condition in which there are no sperm in the ejaculate (semen) [Treatment of female and male infertility. Assisted reproductive technologies. Edited by V.I. Kulakova, B.V. Leonova, L.N. Kuzmicheva. Medical Information Agency. 2005].

Distinguish between obstructive (excretory) azoospermia, in which normally matured spermatozoa do not enter the ejaculate due to the pathology of the vas deferens, and non-obstructive (secretory) azoospermia, in which, due to impaired spermatogenesis, spermatozoa in the testicles mature in small numbers or are absent altogether. It is also possible a variant of the mixed form of azoospermia, associated simultaneously with a violation of both the function of the testicles and the patency of the vas deferens [Infertility in the male. Fourth edition. Edited by L.I. Lipshultz, S.S. Howards, C.S. Niederberger. Cambridge University Press. 2009].

In the case of obstructive azoospermia, mature spermatozoa are often obtained by aspiration from the epididymis [In Vitro Fertilization. Kay Elder, Brian Dale. In Vitro Fertilization. Moscow. MEDpress-inform. 2008].

In most cases, aspiration from the epididymis is performed by simple washing to isolate the sperm. The principle of the method is based on the sedimentation of all cellular elements, including spermatozoa, by centrifugation. The supernatant (supernatant) obtained as a result of centrifugation is removed, and spermatozoa are taken from the resulting sediment for fertilization by intracytoplasmic injection of sperm into the egg - ICSI (intra-cytoplasmic sperm injection) [WHO Guidelines for the Study and Processing of Human Ejaculate. Fifth edition. World Health Organization. "Medical Genetic Research Center" RAS. Capital Print Publishing House, Moscow, 2012].

If a large number of motile epididymal spermatozoa are obtained by aspiration from the epididymis, processing applicable to the native ejaculate (sperm) is allowed, namely density gradient centrifugation. The main criterion for the applicability of this method is a sufficient number of motile sperm. When performing this method, centrifugation of an aspirate from the epididymis in a density gradient containing colloidal beads coated with silane, which separates cells according to their density, is used. In addition, motile spermatozoa actively penetrate the gradient and form a small sediment at the bottom of the tube. The supernatant obtained as a result of centrifugation in a density gradient is removed, and spermatozoa are taken from the sediment formed under the colloidal layer, which can be used for fertilization of oocytes [WHO Guidelines for the Study and Processing of Human Ejaculate. Fifth edition. World Health Organization. "Medical Genetic Research Center" RAS. Capital Print Publishing House, Moscow, 2012]. The density gradient centrifugation method has a limitation on its use in processing material obtained by aspiration from the epididymis: it is not applicable in cases with a small amount of spermatozoa in the material, as well as with low motility or complete immobility of spermatozoa.

In the case of non-obstructive azoospermia, according to published data, no more than 50% of patients have sperm in the testicular tissue. In these cases, a favorable prognosis is possible for obtaining testicular spermatozoa by surgery, while the likelihood of successful treatment depends on the initial cause of the testicular dysfunction (spermatogenesis) and on the practical possibility of isolating testicular spermatozoa in a specific clinical case, since with a small (single amount) and use in IVF (in vitro fertilization) in some cases is impossible, even despite their presence in the resulting material [G. Corona et.al., Sperm recovery and ICSI outcomes in men with non-obstructive azoospermia: a systematic review and meta-analysis. Hum Reprod Update. 2019 Nov 5; 25 (6): 733-757. doi: 10.1093 / humupd / dmz028].

Testicular tissue can be obtained with an open testicular biopsy with or without microdissection, or with a subcutaneous biopsy with a needle [In Vitro Fertilization. Kay Elder, Brian Dale. Ectracorporeal fertilization. Moscow. MEDpress-inform. 2008]. Testicular samples are always contaminated with large numbers of non-spermatogenic cells and red blood cells, often with spermatogenic epithelial cells, as well as cellular debris, which requires additional steps to isolate the sperm. So, for the isolation of germ cells from the convoluted seminiferous tubules testicles at the initial stage of processing to obtain a homogeneous suspension of cells, enzymatic (incubation of a biopsy sample with the collagenase enzyme) or mechanical (grinding) methods are used.

Further processing of the cell suspension is carried out by the method of simple washing by centrifugation, followed by the search and extraction of spermatozoa from the obtained sediment from erythrocytes, non-spermatogenic cells, cells of spermatogenic epithelium and cellular debris [WHO Guidelines for the Study and Processing of Human Ejaculate. Fifth edition. World Health Organization. "Medical Genetic Research Center" RAS. Capital Print Publishing House, Moscow, 2012].

Thus, the current approaches to the isolation (obtaining) of epididymal or testicular spermatozoa from aspiration / biopsy material are based on the centrifugation method with obtaining a cell sediment, in which spermatozoa (often single) are among the predominant mass of other cellular elements, which physically complicates and in some cases it makes it impossible to search for and retrieve spermatozoa (especially in cases of initially small numbers). Therefore, even if there are sperm in the resulting tissue sample, it is not always possible to retrieve and use them for ICSI. Thus, the effectiveness of conventional methods for obtaining spermatozoa from epididymal or testicular tissue is limited by the minimum sufficient number of spermatozoa in the sample, which makes it possible to search for and physically isolate individual germ cells from the sediment after centrifugation.

From the patented methods related to the extraction of sperm, a method is known for removing the human immunodeficiency virus from the sperm of a man. The method provides for the separation of motile spermatozoa from seminal plasma and associated cells by centrifugation and subsequent incubation in special media. According to the invention, sperm is centrifuged in a density gradient (Sil-Select Plus) at 300 g in solutions for 20 minutes, then the supernatant is removed. The sediment containing spermatozoa is aspirated with a pipette, FertiCult Flushing medium is added and centrifuged at 300 g for 10 minutes, after which the supernatant is removed to sediment. Next, the sperm sediment is covered with 2 ml. FertiCult Flushing medium, the tube is placed in an incubator for 80-90 minutes, the most motile spermatozoa move into the medium, they are aspirated in a volume of 0.7 ml. [RF patent 2526494 IPC S12N 7/00, publ. 08/20/2014]. This technique is applicable to the processing of sperm and the isolation of sperm from it, washed from the virus, and includes a combination of conventional semen processing methods: density gradient centrifugation and simple washing, followed by independent ascent of motile sperm from the sediment. The method is applicable to the processing of sperm with relatively normal characteristics, which contain motile spermatozoa that are capable of floating on their own in sufficient quantity for their release. The method is not applicable to sperm with reduced concentration and motility, as well as to aspiration / biopsy material.

A known method of preparing sperm for intrauterine insemination [RF patent 2317072 IPC A61K 31/02, A61P 15/08, publ. 02/20/2008] by taking semen, adding saline and centrifugation twice for 10 minutes at 1500 rpm. After centrifugation, perftoran is injected into the resulting mass of spermatozoa in a ratio of 1: 3, kept for 3-5 minutes, and the insemination procedure is carried out. This method uses the method of simple washing of sperm with subsequent independent emergence of motile spermatozoa from the sediment. The method is applicable to the processing of sperm with relatively normal characteristics, which contain motile spermatozoa that are capable of floating on their own in sufficient quantity for their release. The method is not applicable to sperm with a pronounced decrease in concentration and motility, as well as to aspiration / biopsy material.

The technical problem solved by the present invention is the separation of spermatozoa, including immotile ones and with their critically small (single) amount, from the mass of non-spermatogenic cells, erythrocytes, spermatogenic epithelial cells and cellular debris obtained by aspiration and / or biopsy from the epididymis and / or testicles with the possibility of their subsequent use for fertilization of oocytes and / or cryopreservation.

The technical result consists in obtaining (isolating) a pure fraction of spermatozoa (including single ones) from an epididymis aspirate or a testicular biopsy sample, which can be used for fertilization of oocytes by the ICSI method and / or cryopreservation, including when they are critically small in number, when sperm extraction is standard methods based on obtaining a cell pellet during centrifugation are not effective.

To achieve the above technical result in a method for isolating spermatozoa from aspiration and / or biopsy material from the epididymis and / or testis, the separation of spermatozoa from other cellular elements is carried out by at least two centrifugation cycles, with the selection for each subsequent centrifugation cycle obtained as a result the previous cycle of centrifugation of the supernatant, centrifugation is carried out at 200 - 350 g for 1-2 minutes, cycles of centrifugation of the supernatant are carried out until there is no visible precipitate.

Material for the search and isolation of spermatozoa is obtained by aspiration or biopsy from the epididymis or testicle. Before centrifugation, the biopsy material is pretreated by a mechanical or enzymatic method to obtain a cell suspension. Preferably, centrifugation is carried out at 280-320 g for one minute with a material volume of 8-12 ml. The average number of successive centrifugation cycles is from 5 to 12. With each subsequent cycle, the centrifugation parameters (centrifugal force and time) can be saved or increased relative to the first cycle. Cycles of centrifugation are carried out until there is no visible precipitate, and in most cases a clear supernatant is obtained. Further, if it is necessary to concentrate the obtained fraction of spermatozoa in a minimum volume, the supernatant obtained in the last cycle is centrifuged at 250-350 g for 5-10 minutes to obtain a concentrated fraction of isolated spermatozoa.

The proposed method makes it possible to isolate even single male germ cells from the aspiration / biopsy material and use them for fertilization, especially when the number is critically small, when conventional approaches may be ineffective.

The proposed method is based on the method of differential centrifugation, which has never been previously used in the work with human male germ cells, including when they are obtained from the epididymis or testis.

Differential centrifugation is a widely used method for the isolation and purification of various biological objects, such as viruses, cells, subcellular organelles, proteins and nucleic acids, which are dissolved or dispersed in biologically relevant solvents [De Duve, C. Exploring cells with a centrifuge. Nobel Lecture. 1974, http://www.nobelprize.org/nobel_prizes/medicine/ laureates / 1974 / duve-lecture.html.].

Centrifugation causes precipitation of particles that are heavier than the solvent. The initial solution usually contains a multicomponent mixture of particles that differ in size and density, and, consequently, in their sedimentation rate. The method of differential centrifugation allows you to selectively precipitate the components of interest as a result of repeated centrifugation cycles - in a centrifugal field, all particles (in this case, cells) settle in different ways: large ones are faster than small ones. At the bottom of the tube, as a result of centrifugation, a precipitate, or sediment, of particles is formed. The liquid above the sediment is called the supernatant, and a fairly pure fraction of the smallest particles can be collected from it. Thus, cells that have a sufficient difference in size and sedimentation rate can be separated from each other, while a certain combination of relative centrifugal acceleration and centrifugation time is required, effective to isolate the components of a given multicomponent mixture [Ohlendieck, Kay; Harding, Stephen E. (19 April 2018). "Centrifugation and Ultracentrifugation". Wilson and Walker's Principles and Techniques of Biochemistry and Molecular Biology: 424-453. doi: 10.1017 / 9781316677056.014. ISBN 9781107162273].

The principle of differential centrifugation in the invention is for the first time applied to the isolation of spermatozoa from testicular or epididymal tissue: spermatozoa are smaller in mass than other cells, in the predominant amount present in the biopsy / aspiration material (erythrocytes, non-spermatogenic cells and cells of spermatogenic epithelium), therefore they can be effectively separated from these cells. As a result of differential centrifugation with an experimentally selected centrifugation mode for a given cell suspension, it is possible to obtain an upper fraction (supernatant) free of other elements (erythrocytes, non-spermatogenic cells, cells of spermatogenic epithelium and cellular debris), but containing spermatozoa.

The proposed differential approach in the isolation of male germ cells is much more effective than the traditional method of centrifugation (simple washing), since it avoids the sediment in which spermatozoa are lost or stick, which makes their subsequent isolation in some cases impossible due to their critically small number. ...

The invention is illustrated by the following materials:

FIG. 1. Testicular biopsy material after mechanical processing (x200). There is 1 sperm in the field of view (indicated by an arrow).

FIG. 2. Application of simple washing (A) and differential centrifugation (B) methods to the processing of two parts of the same testicular biopsy material.

FIG. 3. Sediment of erythrocytes, cells of spermatogenic epithelium, non-spermatogenic cells and cell debris after simple washing, containing single spermatozoa (x400). There are 6 fields of view with spermatozoa (indicated by arrows).

FIG. 4. Concentrated sperm fraction, free of most of the erythrocytes, spermatogenic epithelial cells and non-spermatogenic cells, obtained as a result of differential centrifugation (x400). There are 6 fields of view with spermatozoa (indicated by arrows).

A method for isolating spermatozoa from aspiration and / or biopsy material from the epididymis (epididymis) and / or testis for use in in vitro fertilization and / or cryopreservation programs is carried out as follows.

The epididymis / testis material obtained by aspiration or biopsy is placed in a washing medium for human gametes based on HEPES-buffer. If necessary, carry out standard mechanical or enzymatic processing of the tissue in order to homogenize it (obtain a suspension).

Aspiration / biopsy material or a cell suspension obtained after standard processing in a washing medium in a volume of 8-12 ml. placed in a test tube for centrifugation with a conical bottom and centrifuged at the given parameters of centrifugal force and time at 200-350 g for 1-2 minutes. The centrifugal force and centrifugation time depend on the initial sample volume, sperm concentration, degree of contamination with non-spermatogenic cells, erythrocytes, and spermatogenic epithelial cells. When choosing the mode of differential centrifugation for each case, take into account that heavily contaminated samples should be brought up to a larger volume (10-12 ml) with a washing medium. The use of centrifugal force below 200 g and / or centrifugation time less than 1 minute is ineffective, since erythrocytes, non-spermatogenic cells and cells of spermatogenic epithelium do not fully settle to the bottom of the tube; application of centrifugal force above 350 g and / or time more than 2 minutes leads to the simultaneous sedimentation of both erythrocytes, non-spermatogenic cells and cells of the spermatogenic epithelium, and spermatozoa, which does not effectively isolate the pure fraction of the latter. So, for the testicular biopsy material obtained with an open testis biopsy with microdissection, after mechanical processing, the combination of the following parameters of sequential centrifugation is optimal: 300 g for 1 minute in a volume of 8-10 ml.

After each centrifugation, the supernatant is transferred to a new tube and re-centrifuged at the initial parameters or at increased centrifugal force and / or increased time, depending on the condition of the sample, while the centrifugation modes should not go beyond the set interval: centrifugal force 200-350 g, time 1 -2 minutes. Repeated rounds of supernatant centrifugation are carried out until a clear (transparent) supernatant is obtained and / or until there is no visible precipitate; the number of rounds of centrifugation varies on average from 5 to 12 and depends on the initial degree of contamination of the sample with erythrocytes, non-spermatogenic cells and cells of spermatogenic epithelium.

The pure (transparent) supernatant obtained as a result of differential centrifugation, which contains spermatozoa, but from which the bulk of other cells have been removed, can be cryopreserved in the resulting volume or used to select spermatozoa from it during fertilization. If it is necessary to obtain a concentrated fraction of isolated spermatozoa, the supernatant is centrifuged at 250-350 g for 5-10 minutes (depending on the sample volume) in order to concentrate them in a minimum volume. The pellet obtained from the last round of centrifugation is a pure concentrated sperm fraction, which can also be used for fertilization of oocytes by the ICSI method and / or cryopreserved for deferred use in the IVF program.

The implementation of the proposed method is confirmed by clinical data. A total of 257 operations were performed to obtain material from the testis / epididymis in patients with azoospermia. 223 patients diagnosed with non-obstructive (secretory) azoospermia (NOA) underwent testicular biopsy with microdissection under the control of an operating microscope (micro-TESE). 34 patients diagnosed with obstructive azoospermia (OA) underwent aspiration / biopsy from the epididymis (epididymis), including under the control of an operating microscope (MESA / micro-MESE).

The resulting material was processed mechanically (with the exception of the epididymis aspirate) and examined under a microscope. When visualizing at least one sperm in the entire sample volume, it was considered that spermatozoa were found.

The detection rate (presence) of spermatozoa in NOA was 39.9%, which is consistent with published data. In the group with NOA, spermatozoa in all cases of detection were presented in small or single numbers.

The frequency of detection (presence) of spermatozoa in OA was 100%. The indicated efficiency of sperm detection in OA is due to preserved spermatogenesis with impaired patency of the vas deferens. The quality of the material in the OA group varied from a high sperm count to a single number.

Isolation of spermatozoa in all cases was carried out by the method of differential centrifugation. In the group with NOA, the efficiency of sperm isolation by the proposed method (with subsequent use for IVF and / or cryopreservation) was 98.8%. The level of sperm extraction from a surgically obtained material comparable in efficiency has never been previously declared by conventional methods. The high efficiency of sperm isolation is due to the peculiarity of the differential centrifugation method: during processing, it is possible to avoid a concentrated cell sediment, from which the isolation of spermatozoa is extremely difficult or technically impossible, especially in cases of a small (piece) number.

Spermatozoa isolated from biopsy / aspiration material by differential centrifugation were used to fertilize spouse's oocytes obtained on the same day (synchronous IVF cycle) and / or cryopreserved for delayed use in the IVF / ICSI program (non-synchronous IVF cycle). The surplus embryos obtained in IVF programs were cryopreserved for delayed use in thawed embryo transfer (FDE) programs.

In the NOA and OA groups, 147 and 47 cycles of ART with embryo transfer, respectively, were carried out, which included three types of programs: synchronous IVF cycle, asynchronous IVF cycle, and thawed embryo transfer (DE) obtained and cryopreserved in the first two types of programs.

There were 47 pregnancies in 88 patients with NOA in 147 ART cycles (52.8% per patient; 31.9% per ART cycle) and 18 pregnancies in 34 patients with OA in 47 ART cycles (52.9% per patient; 38, 3% per ART cycle).

Thus, the proposed method for isolating spermatozoa from aspiration / biopsy material from the epididymis / testis using the differential centrifugation approach has shown high efficiency both in obtaining spermatozoa (including in cases of a single amount) and when using them in ART and cryopreservation programs.

CLINICAL EXAMPLES

Example 1. Patient A., 34, azoospermia, secretory form (NOA). As a result of a testicular biopsy under the control of an operating microscope (micro-TESE), a sufficient volume of material was obtained, in which, after primary mechanical processing, single spermatozoa were visualized (1 per 20 fields of view, x200), Fig. 1. The machined biopsy material was split in two. One part was processed by the traditional method of simple washing, the second part was processed by the method of differential centrifugation, followed by concentration in the minimum volume. As a result of simple washing, a sediment (Fig. 2A) was obtained from erythrocytes, cells of spermatogenic epithelium and non-spermatogenic cells, containing single spermatozoa (1 per 7 fields of view, x400), Fig. 3. As a result of differential centrifugation, the sperm fraction was separated from erythrocytes, spermatogenic epithelial cells and non-spermatogenic cells and concentrated in a minimal volume (Fig. 2B), the sperm count was 2-5 per field of view (x400) (Fig. 4). From the material processed by the method of differential centrifugation (Fig. 4), it was possible to extract spermatozoa for fertilization of the oocytes of the wife of Patient A., Patient A., by the ICSI method in a synchronous IVF cycle (embryos obtained), part of the spermatozoa was cryopreserved. From the sediment obtained as a result of simple centrifugation (Fig. 3), the extraction of spermatozoa for ICSI was extremely difficult and was not carried out, and cryopreservation of this material was not recommended.

Example 2. Patient M., 38 years old, azoospermia, secretory form (NOA). In the anamnesis of Patient M. there are two unsuccessful testicular biopsy operations: spermatozoa are present in the biopsy sample, but, due to their small number, attempts to isolate them by a simple washing method were previously unsuccessful. As a result of another biopsy from the testicle under the control of an operating microscope (micro-TESE), material was obtained in which, after primary mechanical processing, single spermatozoa were visualized (1 per 30 fields of view, x200), the material was processed by the method of differential centrifugation followed by concentration in a minimum volume ... As a result of processing, a sperm fraction was obtained (1 per field of view, x200), sperm were isolated and used to fertilize the oocytes of the wife of Patient M., Patient I., using the ICSI method in a synchronous IVF cycle, and a pregnancy was obtained as a result of treatment.

Example 3. Patient P., 35 years old, azoospermia, secretory form (NOA). Patient P. has a history of testicular biopsy, in which material containing spermatozoa was obtained. A small volume concentrated sample containing spermatozoa was obtained by simple washing. Due to the extremely low concentration of spermatozoa against the background of a high content of erythrocytes and other cells, the material was concentrated to a minimum volume equal to the volume of one cryosol (200 μl); cryopreservation performed. This cryomaterial was thawed and used in the IVF program, the wife did not become pregnant. During the repeated testicular biopsy, the obtained material was processed by the method of differential centrifugation, as a result of which a pure fraction of spermatozoa was isolated in an amount sufficient for use in the IVF / ICSI program and for cryopreservation. The isolated spermatozoa are cryopreserved in eight cryosolomins, the obtained material is stored for use in upcoming IVF programs.

Example 4. Patient A., 28 years old, azoospermia, genetically determined secretory form (deletion of the AZF-locus). Single sperm cells were found in the testicular biopsy. As a result of differential centrifugation, single spermatozoa were isolated and used to fertilize the oocytes of the wife of Patient A., Patient E., by the ICSI method. The resulting embryos were cryopreserved for delayed transfer in the PRE program.

Example 5. Patient S., 42, azoospermia, obstructive form (OA). From the epididymis aspirate by differential centrifugation, a pure fraction of spermatozoa was isolated in an amount sufficient for use in the IVF / ICSI program and cryopreservation. Cryopreservation of the isolated spermatozoa was carried out in 12 cryosolomines. In an asynchronous IVF cycle, the oocytes of the wife of Patient S., Patient S. were fertilized by the ICSI method using thawed material of one cryosol, one embryo out of six obtained was transferred, pregnancy began. Excess embryos are cryopreserved.

Claims (6)

1. A method of isolating spermatozoa from aspiration and / or biopsy material from the epididymis and / or testis for use in in vitro fertilization and / or cryopreservation programs, including the separation of spermatozoa from other cellular elements of the obtained material by at least two centrifugation cycles with selection for each subsequent centrifugation cycle of the supernatant obtained as a result of the previous centrifugation cycle, and centrifugation is carried out at 200-350 g for 1-2 min, supernatant centrifugation cycles are carried out until there is no visible precipitate. 2. The method of sperm isolation according to claim 1, characterized in that centrifugation is carried out at 300 g for 1 min with a material volume of 8-12 ml. 3. The method of sperm isolation according to claim 1, characterized in that the number of successive centrifugation cycles is from 5 to 12. 4. The method of sperm isolation according to claim 1, characterized in that with each subsequent centrifugation cycle, the centrifugal force is increased relative to the first and / or the previous cycle in the range of 200-350 g and / or time for 1-2 minutes. 5. The method of sperm isolation according to claim 1, characterized in that the biopsy material is pretreated by a mechanical or enzymatic method to obtain a cell suspension. 6. The method of sperm isolation according to claim 1, characterized in that the supernatant taken after the last centrifugation cycle at 200-350 g for 1-2 minutes is concentrated by centrifugation at 250-350 g for 5-10 minutes.

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