RU2669027C1 - Method of stimulation of impaired spermatogenesis and synthesis of testosterone using transplantation of the neonatal testicular tissue - Google Patents

Abstract

FIELD: medicine.SUBSTANCE: invention relates to medicine, namely, to urology and andrology, and can be used to stimulate impaired spermatogenesis and to increase the synthesis of testosterone in the presence of androgen deficiency. Method consists in transplanting tissue of neonatal testis of an animal. To do this, the tissue of testicles in the late stages of development or newborn rats are used. After removal of the tissue, it is ground into fragments of 0.5 × 0.5 mm and washed in physiological saline. Immediately thereafter or after cryopreservation, the tissue is transplanted to pubescent male rats under a kidney capsule under the perididymis.EFFECT: method provides persistent engraftment of transplants and growth of transplanted tissue, which leads to increase in concentration of testosterone in blood and stimulation of spermatogenesis in a damaged testicle with a significant economic effect by eliminating the need for stem cells for these purposes.3 cl, 6 dwg, 5 ex, 1 tbl

Description

The invention relates to medicine, namely to urology and andrology, and is aimed at increasing the effectiveness of the treatment of sexual disorders using cell and tissue therapy.

The problem of maintaining men's health, in particular, sexual function, is becoming increasingly important in connection with a change in the lifestyle of the population and the deterioration of the environmental situation. The incidence of erectile dysfunction and male infertility is increasing. Most often, this condition is associated with the development of androgen deficiency (a decrease in the concentration of testosterone and dihydrotestosterone in the blood) due to age, occupational or medical reasons (Ferrini M.G., Gonzalez-Cadavid NF, Rajfer J. Aging related erectile dysfunction-potential mechanism to halt or delay its onset. Transl. Androl. Urol. 2017 Feb; 6 (1): 20-27. doi: 10.2103 / tau.2016.11.18). The percentage of infertile couples in which infertility is caused by impaired spermatogenesis reaches 15-20 % (Punab M., Poolamets O., Paju P., Vihljajev V., Pomm K., Ladva R., Korrovits P., Laan M. Causes of male infertility: a 9-year prospective monocentre study on 1737 patients with reduced total spermcou nts. Hum. Reprod. 2017. Jan; 32 (1): 18-31. Tyuzikov IA Metabolic syndrome and male infertility (literature review). Andrology and genital surgery. 2013; 2: 5-10). The reasons for the deterioration of spermatogenesis can be different - genetic disorders, cryptorchidism, previous infectious diseases, toxic effects, radiation, chemotherapy, etc., but in the end they come down to 2 pathological changes: blocking sperm maturation at different levels and impaired testosterone synthesis with the development of androgen insufficiency.

The most common method for treating erectile dysfunction and spermatogenesis disorders is hormone therapy using various testosterone preparations of different durations of action (Davidiuk AJ, Broderick GA Adult-onset hypogonadism: evaluation and role of testosterone replacement therapy. Transl. Androl. Urol. 2016 Dec; 5 ( 6): 824-833. Doi: 10.21037 / tau.2016.09.09.02. Shoskes JJ, Wilson MK, Spinner ML Pharmacology of testosterone replacement therapy drugs. Transl. Androl. Urol. 2016. Dec; 5 (6): 834-843 . doi: 10.21037 / tau.2016.07.07.10.). However, with prolonged use, the effectiveness of this therapy decreases and often side effects in the form of metabolic disorders and allergic reactions develop, which forces to cancel this therapy (Sperling N. Side effects of erectile dysfunction drug treatment. Urologe A. 2017. Mar 1. doi: 10.1007 / s00120-017-0341-4).

The development of cell technology using stem cell transplantation opens up new possibilities in the treatment of these conditions.

A known method of treating androgen deficiency by transplanting into the testis allogeneic testosterone-producing Leydig cells (Kirpatovsky ID, Dendeberov ES Allotransplantation of cultural neonatal androgen-producing Leydig cells. Bulletin of the Russian Academy of Medical Sciences 1994; 4: 42-46).

Also known is a method of stimulating impaired spermatogenesis and correcting androgen deficiency using intratesticular bone marrow stem cell transplantation (Lue Y., Erkkila K., Liu PY, Ma K., Wang C., Hikim AS, Swerdloff RS Fate of bone marrow stem cells transplanted into the testis: potential implication for men with testicular failure. Am. J. Pathol. 2007 Mar; 170 (3): 899-908) or mesenchymal cells of adipose tissue (Kamalov A.A., Sukhikh G.T., Zaraysky E .I., Kirpatovsky V.I., Okhobotov D.A., Poltavtseva R.A., Kamenskaya K.A., Makarov E.A. Changes in the Leydig cell population against the background of various experimental options th therapy enriched cell cultures. Natural and Technical Sciences. 2010. №4. pp 95-99).

The disadvantages of these methods are the difficulty of isolating stem cells from biological material, the limited number of selected cell mass and the need for long-term cultivation for the multiplication of stem cells in sufficient quantities. The procedure for obtaining stem cells, maintaining their viability, culturing in nutrient media to obtain a sufficiently large amount of cell mass are complex and expensive procedures that require special sophisticated equipment and highly qualified specialists in the field of cell technology. In addition, the number of viable cells and their proliferative potential after transplantation decreases quite quickly, which may be due to a change in their microenvironment (the so-called "niche"), and this can reduce the effectiveness of cell therapy in the long term.

The closest analogue to the claimed method (prototype) is a method of stimulating spermatogenesis and testosterone synthesis by injection of cultured human testicle cells into an injured testicle (Kamalov A.A., Sukhikh G.T., Kirpatovsky V.I., Zaraysky E.I. ., Poltavtseva R.A., Plotnikov E.Yu., Kudryavtsev Yu.V., Efremov EA, Okhobotov DA Features of testicular tissue regeneration and restoration of fertility in rats on the background of xenograft enriched stem and progenitor cell cultures with bilateral abdominal riptorhizme // Urology. 2008. №6. pp 4-7.). The introduction into the testis with impaired spermatogenesis (an experimental model of abdominal cryptorchidism) of the testicular cell culture of human fetuses or stem cells of the bone marrow led to the regeneration of spermatogenic epithelium and improved male fertility. The disadvantages of this method are the difficulty of obtaining cellular material, its small amount, which requires prolonged cultivation to obtain cell mass, the need to use expensive reagents and equipment. In addition, the necessary therapeutic effect develops slowly - within 3 or more months. A significant drawback of the method is also the mild effect of transplanted stem cells on the restoration of reduced testosterone synthesis by Leydig cells.

The aim of this invention is to increase the efficiency of correction of spermatogenesis disorders and androgen deficiency.

The goal is achieved by using freshly removed or cryopreserved tissue of the neonatal testicle of the fetus or newborn containing a large number of stem cells, and its transplantation into the damaged testicle or under the capsule of the kidney, which allows you to save all the positive effects of cell therapy while minimizing the negative effects of isolation of stem cells from their microenvironment and material costs, and also accelerate the onset of the stimulating effect. The high regenerative potential of the cells of the transplanted tissue contributes to its engraftment and proliferation of cells, which leads to a progressive increase in the mass of the graft, the number of seminiferous tubules, and newly formed Leydig cells. The preservation of the microenvironment (“niche”) of stem cells also contributes to this. As a result, a new anatomical structure is formed that is histologically identical to the tissue of the testis. At the same time, proliferation factors secreted by graft cells contribute to the restoration of the spermatogenic epithelium and Leydig cells and their ability to synthesize testosterone, which restores androgenic status and also stimulates spermatogenesis. The therapeutic effect not only persists for a long time, but also improves as the mass of the implant increases. A persistent engraftment is also due to the fact that the neonatal tissue is transplanted to immunologically privileged places - under the capsule of the kidney and under the white membrane of the testicle, which significantly reduces the likelihood of rejection even with pronounced antigenic differences between the graft and the host body.

The inventive method is as follows.

In fetuses in the late stages of neonatal development or in newborns, the testes are removed, crushed into 0.5 × 0.5 mm fragments, and after washing in physiological saline, the recipient is immediately transplanted or cryopreserved. When transplanting unconserved tissue of the fetal testicle under the kidney capsule, a kidney is isolated from the fat capsule, a small incision is made in the connective tissue capsule along the outer edge, using a miniature probe with a blunt end, form a channel in the subcapsular space, trying not to damage the kidney parenchyma, and a neonatal fragment is placed in this channel testicles. After that, one seam is applied to the incision of the capsule with an atraumatic absorbable thread.

When a non-preserved tissue of the neonatal testicle is transplanted under the testicle, the scrotum is opened, the testicle is taken out into the wound, a small incision is made of the testis by the squirrel in the avascular zone, through which, using a miniature probe, a channel is formed into which a fragment of the neonatal testicle is implanted. One suture with an atraumatic absorbable suture is applied to the incision of the tunica albuginea.

For cryopreservation, the fragmented tissue of the neonatal testicle is placed in a cryoprotective room temperature solution of the following composition (in mmol / l): sodium - 9.3, potassium - 115, magnesium - 4.7, chlorine - 15, phosphates - 57.6, sulfates - 4 , 7, bicarbonate - 9.3, sucrose - 300. After a 5-minute incubation, the cryoprotectant dimethyl sulfoxide (DMSO) is added to the solution at first to a concentration of 5%, after 10 minutes to 10%, and after 10 minutes to 15%. After a 10-minute incubation, neonatal testicle tissue fragments are placed in a dry plastic tube and transferred to a -90 ° C freezer, where they are stored until transplantation. Before transplanting, tissue tubes are removed from their freezer, placed in a water bath at + 37 ° C and after complete thawing (after about 1 minute), they are transferred to a solution similar to cryo-sewn, but with a DMSO concentration of 10%. After 10 minutes of incubation, the tissue is transferred to a solution with a DMSO concentration of 5%, and after 10 minutes to a solution without DMSO. After another 10 minutes, the tissue is prepared for transplantation, which is carried out according to the method described above.

The effectiveness of the proposed method is confirmed by the following examples.

Example 1. Evidence of engraftment of transplanted neonatal testicular tissue in an adult.

Five adult sexually mature male rats underwent transplantation of freshly removed tissue of the neonatal testicle obtained from newborn rat pups 1-2 days after birth under a kidney capsule according to the described procedure. When examining the operated animals after 1, 3, and 6 months in all cases, it was found that under the renal capsule a newly formed tissue was revealed, which, upon histological examination, turned out to be identical to the testicular tissue. With an increase in the observation time, the mass of the implant increased (Fig. 1A. B), while histological examination showed that immature testicular tissue detected 1 month after transplantation was transformed into normal testicular tissue of the adult body with signs of completed spermatogenesis in the seminiferous tubules. (Fig. 2A. B).

Another 5 rats underwent a similar transplantation of neonatal testicular tissue from newborn rat pups under the albumen of the testis of adult males. When observed after 1, 3, and 6 months, as in previous experiments, a newly formed testicular tissue located under the protein coat was detected (Fig. 3).

This example confirms the persistent engraftment of the transplanted tissue of an allogeneic neonatal testicle in the adult body, both under the capsule of the kidney and under the white membrane of the testicle.

Example 2. Proof of the ability of transplanted tissue of the neonatal testicle to synthesize testosterone when transplanted under a kidney capsule.

Neonatal testicular tissue was transplanted from newborn rats according to the method described above under a kidney capsule to 5 previously castrated male rats, and 5 castrated males served as a control (no transplantation was performed). The dynamic determination of testosterone concentration in the blood of castrated rats significantly decreased from 2.39 ± 0.12 ng / ml to 0.61 ± 0.04 ng / ml and the reduced values remained at this level with slight fluctuations over the entire observation period (up to 6 months). In experiments with neonatal testicular tissue transplantation, after 1 month the testosterone level increased to 0.73 ± 0.02 ng / ml, after 3 months to 1.08 ± 0.09 ng / ml, and after 6 months to 1.72 ± 0 , 14 ng / ml.

Thus, the transplanted tissue of the neonatal testicle can not only survive in the adult body, but also synthesize testosterone, which can compensate for the androgen deficiency of the adult body.

Example 3. Proof of the ability of transplanted tissue of the neonatal testicle to restore impaired spermatogenesis and testosterone synthesis when it is transplanted under the white membrane of the damaged testicle.

In 12 rats, spermatogenesis and testosterone synthesis were disturbed by moving the testes for 3 weeks into the abdominal cavity and then releasing them back into the scrotum (model of the abdominal form of cryptorchidism). In the control series (5 rats) no therapeutic effects were performed. In the experimental series (7 rats), before the cryptorchidized testes were brought down into the scrotum, neonatal testicular tissue was implanted under their albumen.

When studying the state of spermatogenesis in control rats, its expressed violation with a block of germination of germ cells at the level of spermatogonia and spermatocytes of the first order was revealed. In individual tubules, only Sertoli cells were detected (Fig. 4A). When observed up to 6 months, a slight improvement in the state of spermatogenic epithelium was noted. In the experimental series, after 1 month, there was a significant stimulation of spermatogenesis up to the appearance of tubules with completely completed spermatogenesis (up to the stage of spermatozoa) (Fig. 4B). With an increase in the duration of observation, the proportion of such tubules increased.

The concentration of testosterone in the blood of control rats remained sharply reduced over the entire observation period, while in the experimental series after 1 month a tendency to its growth was detected, and after 3 and 6 months its concentration even exceeded normal values (Fig. 5).

This example illustrates the effectiveness of transplantation of allogeneic tissue of the neonatal testicles under the albumen of the damaged testicle to restore impaired spermatogenesis and normalize androgen status.

Example 4. Proof of the effectiveness of transplantation of cryopreserved tissue of allogeneic neonatal testis to compensate for androgen deficiency during transplantation under a kidney capsule.

Five castrated males transplanted cryopreserved tissue of an allogeneic neonatal testicle under a kidney capsule according to the previously described method. Five other castrated males served as control (they did not transplant). Determination of blood testosterone revealed a steady decrease in control rats (from 2.39 ± 0.12 ng / ml to 0.61 ± 0.04 ng / ml), whereas in the group of rats with transplantation it increased to 0 after 1 month , 77 ± 0.02 ng / ml, and after 3 months - up to 1.51 ± 0.05 ng / ml. It should be noted that these values are comparable with the data obtained during the transplantation of non-preserved testicular tissue (see example No. 2).

Thus, the cryopreserved tissue of allogeneic neonatal testicles is also able to survive in the adult body and retains its functional properties, in particular, the ability to synthesize testosterone, increasing its concentration in the blood to a subnormal level, which should be an important factor in restoring impaired spermatogenesis.

Example 5. Comparison of the effectiveness of the proposed method and the prototype method.

In 15 rats, the abdominal form of cryptorchidism was modeled by moving both testes into the abdominal cavity for 3 weeks. At the end of this period, the testes were reduced to the scrotum. At the same time, no manipulations were performed in 5 rats (group 1, control), 5 rats were punctured with a suspension of cultured cells of the testes of human fetuses 16-18 weeks of gestation (1 million cells in 0.1 ml of saline solution in each testicle ) (2nd group, prototype method), and another 5 rats under the albumen through a small incision were placed the tissue of the testis taken from newborn rat pups (1-2 days after birth) (3rd group, the claimed method). After 1 month, testicular mass, testosterone concentration in the blood were determined, and histological examination of the testicles was carried out, assessing the state of spermatogenic epithelium and the number of Leydig cells.

The results of the study showed that in the control group (group 1, without treatment) in the vast majority of tubules there was a pronounced violation of spermatogenesis with the blocking of maturation of germ cells at the level of spermatogonia and spermatocytes of the first order and the presence of many tubules completely devoid of spermatogenic epithelium. Only in single tubules did spermatogenesis persist (Fig. 6A). In the 2nd group (prototype method) after 1 month, partial restoration of spermatogenesis was noted, but only up to the stage of spermatocytes of the 2nd order (Fig. 6B). In the 3rd group (the claimed method), the restoration of spermatogenesis was more complete. In many tubules, complete spermatogenesis with sperm detection in their lumen was observed (Fig. 6B).

Moreover, in the 3rd group, testicular mass was more significantly restored. If without therapy, the testicular mass after a 3-week stay in the abdominal cavity and 1 month after being reduced to the scrotum decreased by about 2.5 times, then in experiments with neonatal testicular cell culture (group 2, prototype), their mass exceeded the values obtained in the control series by 28%, and in experiments with neonatal testicular tissue (group 3, the claimed method) - by 78% (table). This indicated a more pronounced testicular regeneration when using the proposed method.

Also in the 3rd group, the proportion of tubules deprived of the epithelium decreased to a greater extent. If in the 2nd group it decreased by about 2.5 times, but still amounted to a significant percentage, then when using the proposed method, such tubules were detected extremely rarely, amounting to only 1.5-4.5% of the total number of tubules in the histological preparation (table).

An important advantage of the proposed method is a more complete restoration of the hormone-producing function of the cryptorchidized testicles. In control experiments, the concentration of testosterone in the blood decreased almost 2 times. In the 2nd group (prototype), the hormone level increased by 69%, while in the 3rd group (the claimed method) it increased by 115% and completely normalized. A more significant stimulation of testosterone synthesis in the 3rd group correlated with the degree of recovery of the Leydig cell population producing testosterone. In the control group, the number of Leydig cells per 1 seminiferous tubule decreased by more than 10 times. In the 2nd group (prototype) the population of these cells increased 6 times, and in the 3rd group (the claimed method) - 9 times, approaching normal values (table).

Thus, the claimed method provides a more complete regeneration of the damaged testicle than traditional stem cell therapy, with pronounced stimulation of spermatogenesis, up to the stage of sperm, and normalization of blood testosterone due to the regeneration of Leydig cells.

The presented examples illustrate the high efficiency of the proposed method. Transplantation of neonatal testicular tissue promotes the formation of a new testicular tissue, with a high ability to synthesize testosterone and stimulate impaired spermatogenesis in the damaged testicle. If there is a need to only correct androgen deficiency, it is possible to transplant neonatal tissue under the kidney capsule. The immune privilege of this zone ensures the engraftment of even allogeneic tissue without additional immunosuppressive therapy, which is extremely important for testicular tissue highly sensitive to the possible toxic effects of pharmaceuticals. Her engraftment ensures the restoration of the Leydig cell pool and their synthesis of testosterone, which eliminates the androgen deficiency.

If there is a need not only to stimulate the synthesis of testosterone, but also to activate impaired spermatogenesis, then neonatal testicular tissue can be transplanted under the white membrane of the damaged testicle. This zone is also immuno-privileged due to the presence of a hemato-testicular barrier, which ensures engraftment of allogeneic tissue. Proliferation of stem cells in a preserved microenvironment (“niche”) leads to the formation of new seminiferous tubules with normal spermatogenesis. In this case, spermatogenic epithelium is regenerated in the surrounding tissue of the damaged testicle, as well as Leydig cells in the interstitium. All this contributes to the normalization of spermatogenesis and hormonal levels.

In addition to the biological effect, the claimed method leads to a pronounced economic effect, since it eliminates the need for isolation and cultivation of stem cells, which requires expensive imported equipment and supplies.

Claims (3)

1. A method of stimulating spermatogenesis and synthesis of testosterone, which consists in transplanting tissue of a neonatal testicle of an animal, characterized in that the tissue of the testicles of the fetus is used in the late stages of development or in newborn rat pups, crushed after removal into 0.5 × 0.5 mm fragments and washed in saline, and transplanted to adult male rats immediately after or after cryopreservation. 2. The method according to p. 1, characterized in that the transplantation is carried out by neonatal testicular tissue under the capsule of the kidney. 3. The method according to p. 1, characterized in that the transplantation of neonatal testicular tissue is performed under the testicle.

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