FURTHER DEVELOPED DEVICE FOR HUMAN SPERM FREEZING BY THE TWENTY-MINUTE METHOD

Transcription

1 FERTILITY AND STERILITY Copyright < 1978 The American Fertility Society Vol. 29, No.3, March 1978 Prinf d in U.S.A. FURTHER DEVELOPED DEVICE FOR HUMAN SPERM FREEZING BY THE TWENTY-MINUTE METHOD JOSEPH BARKAY, M.D.* HENRYK ZUCKERMAN, M,D, Department of Obstetrics and Gynecology, Central Emek Hospital, Afula, Israel A further developed, practical device for human sperm freezing is described by which the preparation of sperm and the freezing process are reduced to a simple procedure requiring about 20 minutes. The instrument is based on the principle of evaporation of liquid nitrogen, which acts as a refrigerant for the freezing plate under the control of a thermostat. Rapid-rate freezing can be performed for pellet production and slow-rat freezing for paillettes; a high degree of isolation of samples is maintained by easily interchangeable freezing plates. Typical stages of freezing are accomplished at temperatures of +5 0 C and C, with storage at -196 C. The recovery index is 50% to 70%. The treatment was applied to 56 patients, resulting in 19 normal deliveries, 3 abortions, and 9 current pregnancies. Prior to 1974 there were three known methods of freezing and conserving human sperm. The first method involves freezing the sperm in paillettes or ampules which are suspended over vapors of liquid nitrogen. 1 The second method involves the use of Dry Ice, on which semen is frozen in the form of drops which yield frozen sperm pellets. 2 The third method is a slow-rate freezing procedure for which fully automated instruments are required. 3-6 Each of these methods has its advantages and disadvantages. The first (manually suspended) system is not very exact, while the Dry Ice method presents a serious problem of isolating different semen samples. The autmatic installation makes sperm freezing a complicated and expensive operation. In 1974 we developed our easy-to-operate, precise, sperm-freezing instrument for pellets and published preliminary results. 7 At the same time we also made some changes in the protective medium which proved to be efficient in our special freezing system. Positive results gave us the incentive to continue to develop our freezing de- Received June 15, 1976; revised September 25, 1977; accepted October 21,1977. *To whom reprint requests should be addressed. vice,t keeping in mind the original goals of a precise rate of freezing, quick and easy operation of the apparatus, and, above all, sterilization and isolation of the samples. The present version of the apparatus may be used either for rapidrate freezing of pellets or for slow-rate freezing of paillettes. MATERIALS AND METHODS The Cryofreezer (Fig. 1). Illustrated in Figure 2 are the novel features of the apparatus (model CSF-16; Ricor Ltd., Ein Harod Ihud, Israel). The tthe difference between the original model and the improved freezing apparatus is as follows: (1) The flrst model consists of freezing chambers with 30 recesses, and the chambers themselves (where the vapors disperse) are fllled with copper waste. On the chambers of the improved model there are 60 recesses and the vapors disperse into the chambers in a labyrinth system. (2) There are two kinds of freezing plates clamped on the chambers for changing. The flrst kind of plate contains 60 recesses for pellets; the second kind contains 12 slots for straws. (3) the Dewar flask has a lo-liter capacity compared with the 2.5-liter capacity of the original model. (4) The front of the improved model contains a metering valve and a flowmeter which facilitate control of the flow rate-freezing rate. This facility is recommended for the use of straws. 304

2 Vol. 29, No.3 FIG. DEVICE FOR HUMAN SPERM FREEZING of constant use, and refrigerant storage for at least 1 week. The control box (C) regulates the rate of freezing and the temperature and indicates these on the control panel. A controlled flow ofliquid N2 is forced by the pressure in the Dewar flask (B) into the freezing chamber (A), where the freezing plate is cooled. When the desired preset temperature is reached the solenoid valve closes. The flow of refrigerant is manually controlled by a needle valve on the control panel, enabling both rapid- and slow-rate freezing. Two types of interchangeable freezer plates are available (Fig. 3), one with 60 recesses to produce pellets of about 0.1 to 0.15 ml each, the other with 12 slots for freezing in straws (paillettes) of 0.5 ml each. The freezing plates are easily removed from the chamber for cleaning, isolation, and sterilization, and they are specially coated to prevent sticking after freezing. On the control panel are a thermometer for indicating and regulating the refrigerant gas, a flow meter, and the Dewar pressure gauge. The entire operation is actuated by means of three pushbuttons-"main", "pressure," and "cool." Presetting the freezer to any temperature between + 5 C and -120 C and maintaining this temperature are achieved with great accuracy by the control circuits. Protective Medium. The composition of the protective medium has remained unchanged since our preliminary report.7 The ratio between semen volume and volume of protective medium is 1:0.66. The protective medium is composed as follows: sodium nitrate (3%), 4.0 ml (40%); glycerol, General view of the sperm cryofreezer. freezing chamber (A) rests on a stainless steel, pressurized liquid nitrogen (LN 2 ) container (B). It holds 10 liters, which last for many hours c esf-i. ''''... 15) LEGEl'W' <\ - Freezing Cha mber n. LN2 f)ewar (' - Control Rox r. - Pressure Gauge N - "Jeedle Valve sv- Solenoirl V<l.lve TC- Temperature Controller F M- 305 r.8.s Flow \leter FIG. 2. Schematic diagram of the sperm cryofreezer.

3 306 BARKA Y AND ZUCKERMAN FIG. 3. Shown are the freezer plates of the sperm cryofreezer with 60 recesses to form sperm pellets and with 12 slots for freezing in straws. ml (5%); egg yolk (fresh), 4.0 ml (40%); crystalline penicillin, 100,000 units in 0.1 ml; streptomycin, 0.2 mg in 0.05 ml. Preparation for Freezing. In order to simplify the procedure we prepare the protective medium as recommended by Bregulla,8 i.e., the protective medium is stored in plastic syringes in a regular freezing box at -20 C. When a quantity of the medium is needed it is thawed in a 36 C water bath together with the semen, in separate test tubes, so that the temperature of both materials will be the same. The next step consists of mixing the protective medium drop by drop with the semen. This step is carried out carefully in order to avoid the destructive effect of the glycerol on the sperm. The mixture is then inserted into a bottle of water at 15 C, two or three ice cubes are added, and the bottle is placed in an ordinary refrigerator for 15 minutes. This has proved to be the optimal time needed to achieve a temperature of +5 C. During this time the thermostat of the control box is set at -80 C; 2 minutes later, when the freezing plate has reached this temperature, the mixture is placed by means of a pipette into the recesses. Within 60 seconds frozen pellets in a volume of 0.1 to 0.15 ml are obtained. March 1978 Storage of Sperm. The frozen sperm pellet is easily removed from the freezing plate with the aid of a plastic spoon and transferred to a test tube containing liquid nitrogen, which lowers the temperature to -196 C. It is very important to keep the level of nitrogen high enough to cover the pellets at all times. This ensures stability of temperature and applies also to the pellets stored in the liquid nitrogen container, where they can stay for decades 9 and still preserve the motility required of the sperm. For this purpose each liquid nitrogen container we use for banking has a special apparatus which indicates when the nitrogen layer has fallen below the required level (model CLD 14 LN2 Low Level Alarm; Ricor Ltd.). Insemination. The patient is inseminated on 4 consecutive days, starting 1 day before the estimated time of ovulation. (Empirical experience has proven that frozen sperm are efficient for no more than 24 hours, whereas fresh sperm may be used for fertilization for 2 or 3 days.3, 10) The patient is placed in an anti-trendelenburg position when the cervical score l l is suitable (spinnbarkeit, ferning, quantity of cervical mucus, and opening of the cervical canal), and the discharge is wiped from the cervix. Five sperm pellets are removed from storage (each pellet contains 4,6 to 7 million spermatozoa*), placed in a dry test tube, and submerged in a 36 C water bath for 2 minutes. After thawing, 0.5 ml of semen is obtained which is injected intracervically until the sperm bubble appears on the cervix, according to the technique of WasterlingY A gum-covered gauze tampon is inserted to prevent the vaginal walls from wiping away the sperm bubble; the tampon is left in place for 10 hours. The quantity of semen inserted is not the determinant of its ability to fertilize. Thus it has been found that, instead of thawing five pellets of semen, a very simple method-the so-called "cold insemination by Bregulla"--can be effected by inserting only one frozen pellet directly into the cervical canal and letting it thaw naturally.13 *According to our calculations, 1 ml of optimal donor's semen having a sperm count of 130 million/ml with a motility of about 85% contains approximat~ly million motile sperm. After adding 1.2 ml of semen to 0.8 ml of protective medium (this matches exactly the volume ratio of 1 ml of semen to 0.66 ml of protective medium), the count of motile sperm in the dilution will be 66.3 million/m!. In optimal cases, after freezing and if the recovery index remains in the range of7w" then in 1 ml after thawing there will remain million spermatozoa. Iffor one pellet we use 0.1 ml of semen, one pellet will contain 4.6 million motile spermatozoa; if we use 0.15 ml, one pellet will contain 7 million motile spermatozoa. i! 1 S ~

4 Vol. 29, No.3 DEVICE FOR HUMAN SPERM FREEZING 307 TABLE 1. Outcome of Treatment of Fifty-Six Patients with AID and AIH Using the "Afula" Method of Freezing and Conserving Human Sperm, Delivery Abortion Current pregnancies Total pregnancies Type of No. of % insemination patients No. of Cycles of No. of Cycles of No. of Cycles of No. of Cycles of Pregnancies patients insemination patients insemination patients insemination patients insemination AID AIR Total RESULTS From 1974 to 1976 we treated 56 patients with frozen semen insemination. Artificial insemination donor (AID) was used in 46 women, of whom 27 (58.6%) became pregnant in 165 insemination cycles. There were 18 deliveries, 2 miscarriages, and 7 current pregnancies (Table 1). Of the 10 patients treated with artificial insemination homologous (AIH), there were 4 pregnancies (40% ) in 33 insemination cycles. One of these patients has delivered, one has miscarried, and two patients currently are pregnant. Of our four AIHtreated pregnant patients, the husbands of three had been treated with caffeine because of oligoand asthenospermia; one of the husbands has since reverteq to this condition. Twenty-five patients who are not yet pregnant have hormonal disturbances or partial tubal occlusion. The results of the follow-up on the 19 children are very satisfactory. None of the children shows any defect in development. It must be mentioned that 16 of the children are boys and 3 are girls. We can find no reason for this phenomenon. Because the patients received insemination every day, including the day of ovulation, the theory may be supported that the movement of Y -bearing spermatozoa is more rapid than that of X bearing spermatozoa, which survive longer. Thus, if insemination occurs on the day of ovulation, Y-bearing spermatozoa would be more likely to fertilize the ovum.14 DISCUSSION The purposes of our study were to simplify our freezing methods and to make the operation of the apparatus easier. We tried a series of protective media to determine which was optimal for our freezing method, and we found 7 that a protective medium containing 40% egg yolk damages motility less than do media described in other reports,3-6 in which 20% egg yolk combined with 20% (5%) glucose or 20% (5%) fructose is used. The recovery index (post-thaw motility/prethaw motility x 100) in our study was 50% to 70%. This result is similar to values reported by others.4, 5, 15 There are many advantages to the newly developed freezing device: (1) the apparatus is simple to operate; (2) it offers reliable performance owing to the 10-liter storage capacity of liquid nitrogen in the instrument; (3) it is always ready for operation (the "run-up" time is 3 minutes) and can be used for single samples; (4) it has a temperature control and indications between +5 C and -120 C; (5) the freezing plates (60 recesses for pellets or 12 slots for paillettes) are easily removed without special tools and are interchangeable. This last feature ensures perfect sterility and isolation of samples. Rapid-rate freezing is recommended for the production of pellets; slowrate freezing is preferred for paillettes. Both modes are easily achieved in the instrument. The rate of flow is controlled by a needle valve and flowmeter. The results of AIH treatment with frozen spermatozoa of oligospermic husbands are very discouraging, as are those of treatment by pooling split ejaculates, because sperm motility in these treatments is not improved after freezing. Our realization of this fact led us to attempt to elaborate a method to increase the motility of frozen human sperm by caffeine treatment.16 REFERENCES 1. Sherman JK: Improved methods of preservation of human spermatozoa by freezing and freeze drying. Fertil Steril 14:49, Nagase H, Niwa T: Deep freezing bull semen in concentrated pellet form. In Fifth International Congress on Animal Reproduction and Artificial Insemination Trento, Italy, 4:410, 1964 ' 3. Behrman SJ, Sawada Y: Heterologous and homologous insemination with human semen frozen and stored in a liquid nitrogen refrigerator. Fertil SterilI7:457, Sokol K, Kaden R, Sperling K, Grossgebauer K: Kryospermaforschung. Fortschr Med 27:1021, Bregulla K, Boquoi UM: Studies on storage of human sperm by deep freezing. Arch Gynaekol 212:323, Bregulla K, Sadowski R: Die Insemination Indication Technic, Ergebnisse. Geburtshilfe Frauenh~ilkd 32:802: 1972

5 308 BARKAYANDZUCKERMAN March Barkay J, Zuckerman H, Heiman M: A new practical method of freezing and storing human sperm and a preliminary report on its use. Fertil Steril 25:399, Bregulla K: Personal communication 9. Sherman JK: Synopsis of the use of frozen human semen since 1964; state of the art of human semen banking. Fertil Steril 24:397, Ackerman DR: The effect of cooling and freezing on the aerobic and anaerobic lactic acid production of human semen. Fertil Steril 19:123, Insler V, Melmed H: The cervical score. Int J Gynaecol Obstet 10:223, Wasterling: Personal communication 13. Bregulla K: Aufarbeitung und Aplication von Kryosperma in Pelletform. In Fortschritte der Fertilitiitsforschung III. Berlin, Grosse Verlag, 1976, p Guerrero R: Association of the type and time of ins emination within the menstrual cycle with the human sex ratio at birth. N Engl J Med 291:1056, Sherman JK: Research on frozen human semen. Fertil Steril 15:485, Barkay J, Zuckerman H, Sklan D, Gordon S: Effect of caffeine on increasing the motility of frozen human sperm. Fertil Steril 28:175, 1977