Vitrification with DMSO protects embryo membrane integrity better than solutions without DMSO.

Vitrification has become common for cryopreservation of embryos. However, the most optimal protocol for vitrification is still to be found. Two vitrification protocols with similar osmolarities were compared: Protocol A, containing dimethyl sulphoxide (DMSO), propane-2-diol, and ethylene glycol, and Protocol B, containing propane-2-diol and ethylene glycol. Viability and the importance of specific incubation times for early embryo recovery, survival, and cleavage were studied. For assessment of cryodamage, embryos were labelled with Alexa Fluor 488-conjugated annexin V and propidium iodide. Vitrification studies on early mouse embryos were followed up with studies on human embryos. The two vitrification protocols did not differ in embryo survival rates and were equally efficient in both mouse and human embryo models. Morphological assessment of embryos directly after vitrification was not a useful tool for assessing survival in this study. Extended exposure of embryos with both vitrification protocols showed that the DMSO-containing vitrification solutions did not lead to cell membrane damage and death as quickly as the DMSO-free vitrification solutions. To assess embryo viability, the authors recommend that vitrification of early embryos should be combined with extended culture and assessment of normal blastocyst development before transferring to patients.

The effects of an antiprogestin, mifepristone, and an antiestrogen, tamoxifen, on endometrial 17 beta-hydroxysteroid dehydrogenase and progestin and estrogen receptors during the luteal phase of the menstrual cycle: an immunohistochemical study.

The effects of a progesterone antagonist, mifepristone (RU486), and an estrogen antagonist, tamoxifen, given during the early luteal phase on endometrial 17 beta-hydroxysteroid dehydrogenase (17HSD) and estrogen (ER) and progesterone (PR) receptors were studied. Eleven regularly menstruating women were studied during control and treatment cycles. In the treatment cycle on day LH + 2 (2 days after the peak serum LH concentration), 10 subjects received a single dose of 200 mg mifepristone, and 9 received 2 doses of 40 mg tamoxifen on days LH + 2 and LH + 3. In addition, 4 subjects received 400 mg mifepristone in a separate treatment cycle. 17HSD, ER, and PR were measured immunohistochemically in endometrial tissue specimens taken on days LH + 6 to LH + 8. Blood samples were conducted during control and treatment cycles, and serum estradiol, progesterone, and LH concentrations were quantified by RIA. Administration of mifepristone blocked the induction of 17HSD by progesterone and prevented the expression of 17HSD in gland and surface epithelial cells in 8 patients. In 2 patients, staining of 17HSD was seen during both the control and mifepristone treatment cycles. The higher dose of mifepristone additionally given to four subjects did not block the expression of 17HSD in 2 cases where blocking was observed with the lower dose of mifepristone, and in 1 of these patients, very strong staining of 17HSD was observed in basal cells beneath the epithelial cells. ER and PR showed intense staining in the nuclei of both gland and stromal cells in mifepristone treatment cycles, whereas receptor staining was faint or absent in the respective control cycles. Tamoxifen did not have any significant effect on staining of 17HSD or the abundance of receptors. Serum concentrations of estradiol, progesterone, and LH were not significantly affected by the administration of mifepristone or tamoxifen. This study reveals that mifepristone, administered in the early luteal phase, usually blocks the expression of 17HSD and the down-regulation of PR and ER. However, the expression of 17HSD in some patients may reflect the ineffectiveness of the mifepristone treatment used to prevent implantation in certain subjects.

Immunization of mice and rabbits by intrasplenic deposition of nanogram quantities of protein attached to Sepharose beads or nitrocellulose paper strips.

Two novel immunization methods designed for immunization with small quantities of antigen, immobilized on a solid matrix and without the use of adjuvant, are presented. The major test antigen used in order to evaluate these methods was bovine serum albumin (BSA). It was deposited in the spleen of mice and rabbits, either attached to Sepharose beads (Pharmacia Sepharose 4B) or to nitrocellulose (NC) paper strips (Millipore). BSA was attached to NC by direct application or by electroblotting after SDS-polyacrylamide gel electrophoresis. The antibody response in mouse and rabbit serum, after intrasplenic immunizations with various quantities of antigen, was analyzed in an ELISA standard procedure. In mice, an antibody response in serum was detected after three intrasplenic immunizations with a total quantity of 73.6 ng BSA bound to Sepharose beads and after two immunizations with a total quantity of 800 ng BSA attached to NC. Determination of the antigen-binding to NC and the clearance rate of antigen attached to NC deposited in the spleen of mice was performed with 125I-labeled BSA. In rabbits, an antibody response in serum was detected after a single intrasplenic immunization with 2.6 micrograms BSA attached to NC. When testing human insulin and sheep prolactin, attached to NC, as antigens in intrasplenic immunization of rabbits, an antibody response was found after a total quantity of 3.2 micrograms insulin and 10.5 micrograms prolactin, respectively.

Intrasplenic immunization for production of monoclonal antibodies against mouse blastocysts.

Applying the intrasplenic immunization method monoclonal antibodies were raised against trophectoderm of mouse blastocysts. Adhesive C57BL/6 blastocysts, obtained 18 h after estrogen reactivation from an experimental delay of implantation, and irradiated with 5000 rad were used as immunogen. Male DBA/2 mice were immunized by four intrasplenic depositions of about ten blastocysts each. The sensitized spleen cells were fused with mouse plasmacytoma cells on the 5th day after the last booster, followed by isolation of hybridoma clones by conventional monoclonal antibody procedures. 82 hybridoma clones were obtained of which two produced IgM antibodies recognizing trophoblast determinants. Absorbing the monoclonal antibodies with C57BL/6 splenic leukocytes followed by immunolabelling of blastocysts demonstrated that the antibodies recognized neither MHC nor TLX antigens. Pre- and peri-implantation stages were mapped by indirect immunofluorescence microscopy. Morulae were negative while blastocysts were positively labeled. Adhesive blastocysts labeled more strongly than delayed blastocysts. Cultured blastocysts showed an intense labeling of some of the trophoblast cells, while other trophoblast cells were unlabeled.

Factors of importance for the establishment of a successful program of intracytoplasmic sperm injection treatment for male infertility.

OBJECTIVE: To establish an intracytoplasmic sperm injection treatment program for couples with male infertility and to determine those factors important for success. DESIGN: A retrospective analysis of 171 consecutive cycles of intracytoplasmic sperm injection concerning 145 infertile couples. SETTING: Infertility clinic in a private hospital associated with a university hospital. PATIENTS: Couples with infertility in the male partner whose sperm parameters were unacceptable for conventional IVF or in whom fertilization by conventional IVF failed repeatedly. INTERVENTIONS: One hundred seventy-one transvaginal oocyte retrievals were completed after superovulation with GnRH agonist and gonadotropins. MAIN OUTCOME MEASURES: The parameters evaluated included fertilization, cleavage, implantation, pregnancy, and spontaneous abortion in relation to patient indications and improved procedures. RESULTS: After intracytoplasmic sperm injection, normal fertilization occurred in 45% of the oocytes (n = 1,499). Of 171 treatment cycles, 93% of the couples had fertilization and 86% had ET. Thirty-six pregnancies were achieved. During the period studied, the mean fertilization rate increased from 21.3% during the first 17 weeks to 67.8% during the last 13 weeks, and the pregnancy rate (PR) per started cycle increased from 12.8% to 31.3%. CONCLUSIONS: Technical factors critical for achieving high rates of fertilization and pregnancy were the use of standardized intracytoplasmic sperm injection pipettes, the immobilization of sperm before injection, and the aspiration of a minimal amount of ooplasm before reinjection with the sperm. Intracytoplasmic sperm injection appears to be superior to other micromanipulation methods for alleviating male infertility.

Platelet-derived growth factor is detected in human blastocyst culture medium but not in human follicular fluid--a preliminary report.

The content of PDGF in human blastocyst culture medium (n = 8), serum (n = 12), and FF (n = 17) from natural IVF cycles was determined by an RIA specific for PDGF B-chain. The blastocysts were cultured under serum-free conditions throughout development. The findings show that PDGF B-chain is released into the culture medium of human blastocysts and that serum is positive, whereas FF is negative for PDGF.

Microfertilization techniques--the Swedish experience.

Intracytoplasmic sperm injection (ICSI) has been studied in this animal research programme since 1990. In 1993, the technique was first applied clinically and up to the present time (September 1994), a total of 456 couples have been studied in 538 cycles. The principal indication for the use of ICSI has been severe male sub-fertility as judged by a semen analysis. In addition, men with high titres of antisperm antibodies, blockage of the vas deferens and neurological disorders such as spinal cord lesions have been included in the programme. Men with genetic disorders such as cystic fibrosis and acrosome-deficient spermatozoa have also been treated successfully. The overall fertilization rate using ICSI was 59%, which is similar to the conventional in vitro fertilization (IVF) programme in Göteborg, however, the pregnancy rate per embryo transfer (29%) and the ongoing pregnancy rate per transfer (22%) were slightly lower. The total number of pregnancies was 144 with 111 of the pregnancies either ongoing or already delivered. To date, 36 healthy children have been born following 29 deliveries and no major malformations have been diagnosed. Being the first programme in Scandinavia to perform ICSI, this unit has experienced long waiting lists which indicates that severe male sub-fertility will be one of the major groups for treatment with assisted reproductive technologies in the future.

Effect of antihormones on endometrial receptors and protein secretion.

The antiestrogen tamoxifen and the antiprogestin RU 486 both interact with respective hormones at the receptor level, RU 486 as a pure antagonist which inhibits endometrial development, the downregulation of estrogen and progesterone receptors and the production of endometrial protein, such as PP14, during the secretory phase of the menstrual cycle. Tamoxifen, on the other hand, has both agonistic and antagonistic action.

The antibody-neutralisation of PDGF, CSF-1, TGFb2,3, EGF and EGF-receptor in utero in pre-implantation mice.

The aim of this study was to determine whether specific growth factors, those shown to be involved using PCR and immunohistochemistry, are necessary for the in-vivo mechanisms of normal implantation in mice. The abdomen of pregnant female mice were opened surgically on day 4 to expose the uterine horns, which were microinjected with specific neutralising antibodies against PDGF, CSF-1, TGFb2,3, EGF and EGF-receptor. At autopsy on day 12, the numbers, positions and sizes of all implantation and resorption sites were recorded. Sham-operation controls were utilised to evaluate the implantation model. Normal female mice exhibited a mean of 6.24 implantation sites per uterine horn. Sham-operated mice exhibited a 30.8% reduction in implantation compared with normals, and saline-injected mice exhibited a 45.7% reduction. Antibody-injected horns were compared with horns injected with saline and horns injected with heat deactivated antibody. All neutralising antibodies tested resulted in significant reductions in the implantation rate and the size of the implantation site. These experiments confirm, in vivo, participation of the specific growth factors tested in the mechanisms of murine implantation, as alluded to previously by evidence from PCR in vitro stimulatory and immunohistochemical work.

Experiments with immunization of mice with blastocysts by an intrasplenic route.

A few attaching blastocysts from CBA/H mice were irradiated from a Cesium source and transferred into the spleen of male DBA2 mice. A booster immunization was performed after four weeks. Blood samples for preparation of antiserum to test for the presence of immunoglobulins directed against blastocyst surface determinants were obtained by a retro-orbital puncture. Specific antibodies were detected with a protein A-gold method, modified for transmission electron microscopy of air-dried blastocysts. The results showed that CBA/H blastocyst incubated in DBA2 immune serum were positive for protein A-gold labelling, while control blastocysts only possessed a few irregularly scattered gold particles. Thus, it seems as a deposition of antigens in the spleen tissue with persistence of the antigens at this site will result in detectable antibodies in the peripheral blood.

Methods for production and detection of monoclonal antibodies against surface components of adhesive implanting mouse blastocysts.

Monoclonal antibodies against mouse blastocysts in the adhesion stage of implantation were obtained by intrasplenic immunization with blastocysts either injected into the spleen after irradiation or transferred after drying on a piece of nitrocellulose paper. About 10 blastocysts were deposited at each of 4 immunizations before the spleen cells were used for hybridoma production. The supernatants obtained were examined for anti-blastocyst antibodies in indirect immunofluorescence labelling of native blastocysts and with ABC-immunoperoxidase staining either of blastocysts attached to nitrocellulose paper or of methanol-fixed blastocysts. Further, sections of paraffin-embedded blastocysts were used for detection of antigens.

Simplification of IVF: minimal monitoring and the use of subcutaneous highly purified FSH administration for ovulation induction.

During the past few years much effort has been put into simplifying the clinical management of in-vitro fertilization/embryo transfer cycles. One important step was the introduction of transvaginal ultrasound-guided oocyte collection, as previously described. This study describes further simplifications of the clinical management of ovarian stimulation. During the period 1st January 1991 to 31st August 1993, three major simplification steps were introduced. All cycles were down-regulated with a gonadotrophin-releasing hormone (GnRH) agonist according to a long protocol permitting fairly precise programming of the oocyte collection. During period I (n = 329 cycles), closer monitoring by several pelvic ultrasound scans and serum oestradiol was used for monitoring the ovarian stimulation. During period II (n = 230 cycles), only one ultrasound scan was used for monitoring the ovarian cycle; oocyte collections during weekends were avoided. During period III (n = 386 cycles), further simplification of the clinical management was introduced by using a highly purified follicle stimulating hormone (FSH) (Fertinorm/Metrodin HP), which was self-administered s.c. for ovarian stimulation. The take-home baby rates per started cycle for periods I, II and III were 16.4, 32.6 and 31.3% respectively. These figures indicate that when using long down-regulation with a GnRH agonist, simplification of the monitoring of the ovarian stimulation is possible without decreasing the pregnancy rate. Furthermore, the use of a highly purified FSH, self-administered s.c., greatly simplified treatment without compromising cycle outcome or increasing the risk of developing an ovarian hyperstimulation syndrome.

Trophectoderm surface expression of the cell adhesion molecule cell-CAM 105 on rat blastocysts.

A variety of cellular interactions is involved in the process of implantation of the mammalian embryo into the uterine tissue. Recent discoveries have demonstrated that intercellular recognition and adhesive events are governed by a class of cell surface molecules known as cell adhesion molecules (CAMs). In the present report, we have investigated the occurrence of the well-characterized cell adhesion molecule cell-CAM 105 on the surface of rat pre- and peri-implantation embryos of various stages. This was carried out by indirect immunofluorescence microscopy employing affinity-purified rabbit antibodies against cell-CAM 105. The embryonal stages investigated comprised morulae, normal day-4 blastocysts, and delayed and adhesive blastocysts obtained by using the method of experimentally delayed implantation. Cell-CAM 105 was absent in the early-morula stage, but in normal day-4 blastocysts and delayed blastocysts a specific staining for cell-CAM 105 was seen on the entire surface. However, adhesive-stage blastocysts exhibited a marked polarity with staining of the polar trophoblast cells. Scanning electron microscopy of adhesive-stage blastocysts revealed that the stronger staining of the polar region was not due to a greater number of microvilli on the polar trophoblast cells. Thus, it seems as if cell-CAM 105 is lost or masked from the surface of the mural trophoblast cells of adhesive-stage rat blastocysts. Since the mural trophoblast cells are the first to adhere to the uterine luminal epithelium during the onset of implantation and subsequently invade the uterine stroma, we suggest that the apparent downregulation of cell-CAM 105 in the mural trophoblast cells might be linked to the acquisition of trophoblast invasiveness.

Mouse blastocyst surface expression of galactose-containing epitopes coinciding with trophoblast differentiation.

A galactose-containing cell surface epitope of mouse blastocysts was identified and partially characterized by means of immuno- and lectincytochemistry, using a mouse IgM anti-blastocyst monoclonal antibody (mAb N63) and four different galactose-binding lectins (BSL-1, DBA, PNA and SBA) as molecular probes. The mAb was produced by syngeneic intrasplenic immunization with adhesive mouse blastocysts, obtained 18 h after estrogen reactivation from facultative delay of implantation. Labelling of different mouse embryonic stages collected by uterine flushings revealed that the labelling of the epitope by monoclonal antibodies was restricted to the blastocyst stage. A peak labelling intensity was observed on late blastocysts. When examining blastocyst outgrowths, both trophoblast and embryoblast were weakly stained by mAb N63. Direct antigen characterization performed on blastocysts indicated that the mAb N63 recognized a galactose-containing glycolipid antigen. Immunochemistry of cryosectioned, unfixed mouse tissues including ovary, testis, uterus in delay and at implantation, Day 12 and term placenta, liver, kidney, brain, intestine, heart, striated muscle, and skin was negative. In addition, labelling of rat and hamster blastocysts was negative. In vitro experiments demonstrated that the galactose-containing blastocyst surface epitope was not involved in blastocyst attachment to plastic culture dishes. The appearance of the epitope at the embryonic surface in vivo coincides with the time of trophoblast differentiation and implantation in the mouse.

The outcome of intracytoplasmic sperm injection is unrelated to 'strict criteria' sperm morphology.

Sperm morphology was assessed according to the 'strict criteria' established for in-vitro fertilization treatment in the semen samples used for 354 consecutive treatment cycles for intracytoplasmic sperm injection (ICSI). The semen samples were classified according to the three predictive categories of the Tygerberg strict criteria: excellent prognosis (>14% morphologically normal spermatozoa), good prognosis (4-14%) and poor prognosis (<4%). It was found that 37 (10.5%) of the ICSI cycles belonged to the excellent prognosis category, 197 (55.6%) to the good prognosis category, and 120 (33.9%) to the poor prognosis category. The outcomes of the ICSI treatments were evaluated and compared with the sperm morphology classification in order to determine whether the strict criteria could aid in predicting the outcome of ICSI. The fertilization rates in the three categories were 61.6, 66.8, and 61.9%, the pregnancy rates per oocyte retrieval 18.9, 24.9, and 28.3%, and the implantation rates 9.9, 13.0, and 14.9% respectively. No significant differences were found in fertilization, pregnancy, or implantation rates between the three prognosis categories, i.e. the poor prognosis category had an equal chance of obtaining pregnancy compared with the good prognosis category. The results indicate that strict sperm morphology is not related to the outcome of ICSI.

Expression of cellCAM-105 in the apical surface of rat uterine epithelium is controlled by ovarian steroid hormones.

Affinity-purified antibodies to cellCAM-105, an adhesive cell surface glycoprotein, were used in immunohistochemical investigations of rat uteri at various functional stages: (i) the oestrous, pro-oestrous, metoestrous, and dioestrous stages of the oestrous cycle, (ii) Days 1-8 of normal pregnancy, (iii) delayed implantation, (iv) 18 h after oestrogen reactivation from delay of implantation, and (v) juvenile rats, and normal ovariectomized adults, respectively, before and after experimental injection of progesterone and/or oestrogen. CellCAM-105 was present in the apical zones of the luminal and glandular epithelium cells in a stage-specific and hormone-dependent manner. The results indicate that: (1) steroid hormones are essential for the expression of cellCAM-105 in the uterine epithelial cells; (2) progesterone induces cellCAM-105 expression in the glandular epithelium, and oestrogen induces cellCAM-105 expression in the luminal epithelium; (3) progesterone induces down-regulation of cellCAM-105 from the surface of the uterine luminal epithelium of juvenile rats; (4) cellCAM-105 is absent in the luminal epithelial cells but present in the glandular epithelial cells of the rat uterus at the time of blastocyst implantation.

Subzonal insemination (SUZI) or in vitro fertilization (IVF) in microdroplets for the treatment of male-factor infertility.

PURPOSE: The results of subzonal insemination (SUZI) and in vitro fertilization with microdroplet insemination used in couples with male-factor infertility are presented. RESULTS: The total fertilization rate was 17.4% for SUZI (n = 89) and 49.3% for microdroplet IVF (n = 100). The fertilization rate for standard IVF (n = 510), not including any male-factor infertility and performed during the same period, was 73.2%. The "take-home baby rate" per started cycle and per embryo transfer (ET), respectively, was 10 and 17.6% for SUZI and 20 and 24.7% for microdrop IVF. For standard IVF these figures were 27 and 31.7%. CONCLUSION: It was concluded that microdroplet IVF can be used with good results in cases of moderate male-factor infertility. The normal (2PN) fertilization rate with the SUZI technique was only 15.1%. However, despite the low fertilization rate, SUZI should be considered when dealing with severe male-factor infertility.

Early luteal phase treatment with mifepristone (RU 486) for fertility regulation.

Mifepristone (RU 486) is an antiprogestin which interacts with progesterone at the receptor level. Administration of mifepristone immediately after ovulation does not upset the menstrual cycle. However, the maturation and function of the endometrium is inhibited and uterine contractility is changed. To test if these effects are sufficient to prevent implantation, 21 women agreed to use one single treatment with 200 mg mifepristone on day luteinizing hormone (LH) + 2 monthly as their only contraceptive method. The women were treated for 1-12 months. The time of the LH peak was determined in the urine by the women themselves using a rapid LH test (Ovu-quick, Organon). The overall number of cycles studied was 169. In 12 cycles the women were unable to detect the LH peak. In these cycles no treatment was given and the women advised to use barrier methods during the time to menstruation. The remaining 157 cycles with a detectable LH peak were all ovulatory based on plasma progesterone measurement. One pregnancy occurred. On the basis of the time of the LH peak, it was retrospectively calculated that in 124 cycles at least one act of intercourse occurred during the period 3 days before to 1 day after ovulation. The probability of pregnancy in this period of the menstrual cycle is thus 0.008. The women did not complain of any treatment-related side-effects apart from slight bleeding for 2-3 days starting a few days after the day of treatment in 35% of the cycles.(ABSTRACT TRUNCATED AT 250 WORDS)

PIP: Mifepristone (RU-486) is an antiprogestin which interacts with progesterone at the receptor level. The objective was to determine whether the effects on endometrial development and function and on uterine contractility of immediate post-ovulatory treatment with mifepristone could prevent pregnancy. 21 fertile, sexually active women with regular menstrual cycles were treated with a single dose of 200 mg mifepristone 2 days after the luteinizing hormone (LH) surge (LH + 2) on a monthly basis for 1-12 months. The time of the LH peak was determined in the urine by the women themselves using a rapid LH test (Ovu-quick, Organon), and this was confirmed later by radioimmunoassay. All the women, except one, had previously had at least 1 delivery and 1 pregnancy terminated. Each woman measured the urine concentration of LH twice daily, starting about 4 days prior to the expected time of ovulation (normally day 10 of the cycle) and continuing until 1 day after the maximum LH concentration. The plasma concentration of progesterone was measured 5 days and human chorionic gonadotrophin (HCG) 2 weeks after the treatment in all cycles. The overall number of cycles studied was 169. In 12 cycles the women were unable to detect the LH peak. The remaining 157 cycles with a detectable LH peak were all ovulatory based on plasma progesterone measurement. 1 pregnancy occurred and was terminated by vacuum aspiration. Based on the time of the LH peak, it was retrospectively calculated that in 124 cycles at least 1 act of intercourse occurred between 3 days before and 1 day after ovulation. The probability of pregnancy in this period of the menstrual cycle was thus 0.008. The were no treatment-related side effects apart from slight bleeding for 2-3 days starting a few days after the day of treatment in 35% of the cycles. The effect of mifepristone on the endometrium was sufficient to prevent pregnancy, therefore it can be used for contraception.