Producing primate embryonic stem cells by somatic cell nuclear transfer.

Derivation of embryonic stem (ES) cells genetically identical to a patient by somatic cell nuclear transfer (SCNT) holds the potential to cure or alleviate the symptoms of many degenerative diseases while circumventing concerns regarding rejection by the host immune system. However, the concept has only been achieved in the mouse, whereas inefficient reprogramming and poor embryonic development characterizes the results obtained in primates. Here, we used a modified SCNT approach to produce rhesus macaque blastocysts from adult skin fibroblasts, and successfully isolated two ES cell lines from these embryos. DNA analysis confirmed that nuclear DNA was identical to donor somatic cells and that mitochondrial DNA originated from oocytes. Both cell lines exhibited normal ES cell morphology, expressed key stem-cell markers, were transcriptionally similar to control ES cells and differentiated into multiple cell types in vitro and in vivo. Our results represent successful nuclear reprogramming of adult somatic cells into pluripotent ES cells and demonstrate proof-of-concept for therapeutic cloning in primates.

Live birth after ovarian tissue transplant.

Radiation and high-dose chemotherapy may render women with cancer prematurely sterile, a side-effect that would be avoided if ovarian tissue that had been removed before treatment could be made to function afterwards. Live offspring have been produced from transplanted ovarian tissue in mice and sheep but not in monkeys or humans, although sex steroid hormones are still secreted. Here we describe the successful transplantation of fresh ovarian tissue to a different site in a monkey, which has led to the birth of a healthy female after oocyte production, fertilization and transfer to a surrogate mother. The ectopically grafted tissue functions without surgical connection to major blood vessels and sets the stage for the transplantation of cryopreserved ovarian tissue in humans.

Isolation and characterization of rabbit endocervical cells.

Three cytologically distinct cell populations were identified, in addition to ciliated cells, when a unit gravity sedimentation procedure was applied to pronase-dispersed rabbit endocervical cells. Two of these cell populations contained histochemically distinguishable (periodic acid- Schiff [PAS]) mucoproteins and were designated vacuolated and granular PAS-positive cells. The third, designated as vacuolated PAS-negative, did not contain secretory granules. Cell integrity was confirmed by trypan blue dye exclusion, [(3)H]leucine incorporation, and ultrastructural analysis. To demonstrate hormonal modulation of endocervical cell morphology, cell distribution profiles were compared from animals in different hormonal states. In the absence of estrogen dominance, PAS- positive cells from 5-d pseudopregnant rabbits were reduced 50 percent, while vacuolated PAS-negative cells increased fourfold as compared with estrous cell populations. The PAS-positive cells sedimented toward the top of the gradient where the bovine serum albumin concentrations were lower, consistent with a reduction in the number of secretory granules. In the sustained absence of ovarian steroid hormones, the number of PAS-positive mucous cells from ovariectomized rabbits was reduced to only 4 percent of the total endocervical cell population. The biosynthetic capacity of isolated endocervical cells was determined by incubating the three nonciliated cell populations from estrous and 5-d pseudopregnant rabbits for 36 h with the mucin precursor, [(14)C]N-acetyl- D-glucosamine. Only PAS-positive cells incorporated significant amounts of labeled precursor. This study indicates that steroid hormones influence cervical secretions by modulating the type of endocervical cells.

Artificial insemination and the assisted reproductive technologies in non-human primates.

The experience with artificial insemination (AI) and the more invasive ARTs (assisted reproductive technologies) in the propagation of non-human primates (NHPs), although limited, has included representation from the Great Apes and both Old World and New World Macaques. The application of these technologies in NHPs is impacted by high cost, substantial technical requirements and the limited captive populations of available animals. A major incentive for their use would be to propagate endangered, underrepresented individuals or valuable founder animals. Detailed protocols and a substantial experience base for the ARTs are available for rhesus and cynomolgus macaques and form the basis of this review, including sperm recovery, processing and long-term storage at low temperatures, insemination techniques and timing. Controlled ovarian stimulation and subsequent oocyte recovery required for the invasive ARTs such as intracytoplasmic sperm injection (ICSI), is also described. Three recent AI reports in Old World Macaques are reviewed, along with examples of the use of the ARTs in the propagation of valuable founder animals, in the preservation of endangered macaques, and finally in the creation of neurodegenerative disease models for biomedical research purposes.

The non-human primate oocyte and embryo as a model for women, or is it vice versa?

The role of the non-human primate (NHP) oocyte and embryo in translational research is considered here including both in vitro activities directly involving oocytes or embryos as well as animal studies that impact reproductive function. Reasons to consider NHPs as animal research models along with their limitations are summarized. A case is made that in limited instances, such as in the development and application of the assisted reproductive technologies or in the study of embryonic stem cells, the human oocyte and embryo have acted as models for the monkey. The development of strategies for the preservation of fertility is used as an example of ongoing research in the non-human primate that cannot be conducted in women for ethical reasons. In animal studies, monitoring reproductive potential, responses to embryonic stem cell transplantation, along with translational research in the field of contraceptive development for women are considered as subjects that benefit from the availability of a NHP model.

Composition and functioin of human cervical mucus.

Human cervical mucus was collected from seven donors during the follicular, ovulatory and luteal phases of the ovulatory menstrual cycle. Individual mucus samples were solubilized and fractionated on Sepharose columns into excluded mucins and low-molecular-weight proteins. Mucin fractions were highly purified, as evidenced by the presence of a single N-terminal amino acid residue, threonine, and by the absence of contaminating plasma proteins. Amino acid compositions of mucins isolated during different menstrual phases of a single donor or from different donors were similar. Mucin carbohydrate compositions were also similar, except for the sialic acid-to-fucose ratio, which varied significantly between donors but not within the menstrual cycle of a single donor. An analysis of variance was applied to evaluate the contribution of mucin composition to viscoelasticity, as quantitated by microrheometry. Viscoelasticity was dependent on the donor, on the percent nondialyzable solids and on the mucin content, b ut not on the phase of the menstrual cycle during which the sample was collected. These findings suggest that mucus function (viscoelasticity) is reflected in carbohydrate composition and/or structure and that this relationship is unique for each donor. Furthermore, the absence of menstrual phase-dependent differences in mucins suggests that mucin concentration and not composition changes in response to alterations in the hormonal milieu.

Effect of mucolytic agents on the rheological properties of tracheal mucus.

Canine tracheal mucus was dissolved by a number of mucolytic agents, including disulfide bond reducing agents, hydrogen bond breaking agents, and chaotropic ions, and their effect on rheological properties was assessed. Sodium thiocyanate led to 85-100% dissolution with the maximum retention of elasticity. Thiocyanate exposure did not result in demonstrable alterations in the size or shape of the mucus glycoproteins. Sodium thiocyanate is therefore recommended as a suitable dispersing agent for physiochemical studies of glycoprotein secretions.

Titrating luteinizing hormone surge requirements for ovulatory changes in primate follicles. I. Oocyte maturation and corpus luteum function.

The amplitude and duration of the midcycle LH surge required for ovulatory maturation of the follicle and its enclosed oocyte in primates are unknown. To titrate periovulatory LH requirements, female rhesus monkeys received human gonadotropins (FSH with/without LH) for 9 days beginning at menses to promote the development of multiple preovulatory follicles. The next day, animals (n = 4-6/group) received: 1) no ovulatory stimulus; 2) 1000 IU hCG, im; 3) one injection of 100 micrograms GnRH, sc (GnRH-1); 4) three injections of GnRH (GnRH-3) at 3-h intervals (0800, 1100, and 1400 h); or 5) two injections of 50 micrograms GnRH agonist (GnRHa), sc, 8 h apart (0800 and 1700 h) to induce ovulatory maturation. Follicles were aspirated 27 h after the hCG or initial GnRH/GnRHa injection or on days 8 and 10 in animals receiving no ovulatory stimulus. Nuclear maturity of oocytes was evaluated as a marker for reinitiation of meiosis. Estradiol and progesterone levels were determined in daily serum samples by RIA. Levels of LH(-like) bioactivity were measured at selected intervals after hCG injection and within 24 h of GnRH/GnRHa treatment. In all groups, estradiol continuously rose to similar peak levels on day 10. The hCG treatment markedly elevated circulating LH-like bioactivity for up to 3 days. In GnRH-1, bioactive LH increased to 433.1 +/- 170.2 ng/mL (mean +/- SEM; n = 3) within 1-2 h, but then decreased to baseline (4.9 +/- 1.5 ng/mL) within 6 h. GnRH-3 and GnRHa treatment extended the interval of elevated bioactive LH to 8 and 14 h, respectively. There was no difference in the peak levels of LH(-like) bioactivity reached after hCG, GnRH, or GnRHa injection. Functional luteal phases were absent in monkeys receiving no ovulatory stimulus, whereas hCG treatment increased progesterone levels to 101 +/- 9 nmol/L (n = 6) and elicited functional luteal phases of 11.8 +/- 0.4 days. In contrast, only one animal in the GnRH/GnRHa groups (i.e. one GnRH-3 monkey) displayed elevated progesterone levels in the luteal phase. Of the total cohort of oocytes aspirated from follicles, a greater (P less than 0.05) proportion were classified as being in metaphase I or II of meiosis after hCG treatment (86%) compared to no ovulatory stimulus (13%), GnRH-1 (0%), GnRH-3 (43%), and GnRHa (12%). Thus, GnRH elicits a transient LH surge that can be extended by GnRH-3 or GnRHa in stimulated cycles of monkeys.(ABSTRACT TRUNCATED AT 400 WORDS)

Administration of human luteinizing hormone (hLH) to macaques after follicular development: further titration of LH surge requirements for ovulatory changes in primate follicles.

After stimulation of multiple follicular development, endogenous LH surges elicited by GnRH or GnRH agonist were of insufficient duration (4-14 h) to evoke oocyte maturation and luteinization in this species. In this study, periovulatory LH surge requirements were further titrated using hLH as the ovulatory stimulus. Beginning at menses, rhesus monkeys were treated with human gonadotropins for 9 days to stimulate follicular growth. To induce ovulatory maturation on day 10, animals received: 1) hCG (1000 IU, im; n = 8); 2) highly-purified, urinary hLH (2542 IU, im; n = 4); or 3) hLH (2542 IU, im) followed by three injections of hLH (200 IU, im) at 8-h intervals (0800, 1600, 2400 h) daily during the luteal phase until menses (n = 3). Oocytes and luteinizing granulosa cells were obtained via follicle aspiration 27 h after the initial hLH or hCG injection. Estradiol and progesterone levels were measured in daily serum samples by RIA. Bioactive LH levels were determined at selected intervals within 36 h of the hLH ovulatory stimulus. Nuclear maturity of oocytes was evaluated as an indicator for reinitiation of meiosis. Luteinizing granulosa cells were processed for indirect immunocytochemistry using a monoclonal antibody to human progesterone receptor. In vitro progesterone production by luteinizing granulosa cells over 24 h was also assessed in the absence and presence of hCG. In all groups, serum estradiol rose to similar peak levels on day 10. After hLH, bioactive LH levels peaked (1262 +/- 79 ng/mL; mean +/- SEM) by 2-6 h, declined thereafter but remained above surge levels (100 ng/mL) for 18-24 h. Within 24 h of hLH injection, serum progesterone increased to 13 +/- 3 nmol/L, but returned to baseline in 1-6 days. In contrast, higher levels of progesterone were observed after hCG (114 +/- 51 nmol/L) and during luteal phase treatment with hLH (137 +/- 25 nmol/L) and the luteal phase was longer (11.5 +/- 0.4 and 14.3 +/- 0.7 days, respectively). Of the total cohort of oocytes aspirated, the proportion of oocytes resuming meiotic maturation (metaphase I plus metaphase II) was similar after hCG (76%) and hLH (74%). However, the proportion of oocytes maturing to metaphase II tended to be less (P = 0.08) after hLH (13%) than hCG (22%). Fertilization rates were similar between the two groups. Progesterone receptor was detected in nuclei of luteinizing granulosa cells from all animals receiving hCG, but only in some given hLH.(ABSTRACT TRUNCATED AT 400 WORDS)

Initiation of periovulatory events in primate follicles using recombinant and native human luteinizing hormone to mimic the midcycle gonadotropin surge.

The amplitude and duration of the midcycle LH surge required for periovulatory changes in the primate follicle are incompletely defined. We reported that short (4- to 14-h) LH surges were insufficient to induce periovulatory events after multiple follicular development in macaques. In contrast, an 18- to 24-h LH surge induced oocyte maturation plus granulosa cell luteinization, but did not support corpus luteum function. In this study, the periovulatory changes following LH surges of 48 h elicited using pituitary (pit) or recombinant (r) human (h) LH were compared to those after 24-h LH surge durations or after urinary hCG (u-hCG) treatment. Beginning at menses, rhesus monkeys were treated with human gonadotropins for 9 days to stimulate follicular growth. On day 10, animals (n = 3-5/group) received 1) a single injection of u-hCG [79 +/- 3 micrograms RP-1 equivalents (equiv), im], 2) two injections of pit-hLH (91 +/- 4 micrograms RP-1 equiv, im), 3) one injection of r-hLH (21 +/- 1 micrograms RP-1 equiv, im), or 4) two injections of r-hLH (21 +/- 1 micrograms RP-1 equiv). Oocytes and granulosa cells were obtained via follicle aspiration 27 h after the initial LH or hCG injection. In all groups, serum estradiol rose to similar peak levels by day 10. Circulating LH-like bioactivity was elevated for more than 48 h after u-hCG. Peak serum LH bioactivities were proportional to the administered LH doses, as determined in the in vitro bioassay. Two injections of either r-hLH or pit-hLH elicited surge levels (> 100 ng/mL) of bioactive LH for 36-48 h, whereas one injection sustained surge levels for only 18-24 h. The proportions of oocytes resuming meiosis (68-76%) were similar in all groups. Immunocytochemical staining for progesterone receptor and in vitro progesterone production by granulosa cells in all LH-treated groups were comparable to those of cells form the hCG-treated group. Peak levels of progesterone in the luteal phase were comparable in monkeys treated with two doses of pit-hLH and r-hLH (18.5 +/- 10.4 vs. 8.1 +/- 1.5 ng/mL) and approached that in u-hCG treated monkeys (39.5 +/- 18.0 ng/mL). However, progesterone levels in animals treated once with r-hLH (3.4 +/- 1.5 ng/mL) were less (P < 0.05) than those in u-hCG-treated monkeys.(ABSTRACT TRUNCATED AT 400 WORDS)

Administration of an aromatase inhibitor during the late follicular phase of gonadotropin-treated cycles in rhesus monkeys: effects on follicle development, oocyte maturation, and subsequent luteal function.

Local modulation of follicular and gametogenic functions by ovarian androgens and estrogens in mammalian species has been proposed. This study examined the effects of elevated androgen/estrogen ratios during follicular maturation in vivo by inhibiting aromatase activity in rhesus monkeys. To obviate steroid feedback effects, gonadotropin-treated animals were used. Beginning at menses (day 1), animals received human (h) FSH (60 IU/day, im) on days 1-6, followed by hFSH plus hLH (60 IU/day, im) on days 7-9 to promote the growth of multiple follicles. Ovulatory maturation was induced by hCG (1000 IU, im) on day 10. On days 8-10, four animals received an aromatase inhibitor, 1,4,6-androstatrien-3,17-dione (ATD; 1-1.25 g, orally, twice/day), while five served as controls and received no further treatment. Within 8 h of ATD treatment, a 63% reduction in serum estradiol levels relative to control values was evident, which reached maximal suppression (84%) by day 10. A marked elevation (17-fold) in serum androstenedione and a lesser increase (2.6-fold) in serum testosterone occurred with aromatase inhibition, yielding androstenedione/estradiol (18.0) and testosterone/estradiol (1.9) ratios greater than those in controls (0.6 and 0.3, respectively). ATD treatment did not alter follicular diameters or the total number of follicles per animal (20 +/- 3) relative to control values (16 +/- 3). Of the total cohort classified, the proportion of oocytes collected at prophase I was greater (P < 0.05) after ATD treatment (31%) than in controls (11%). Completion of oocyte meiosis to metaphase II was retarded (P < 0.05) in ATD-treated (4%) compared to control (26%) animals. Furthermore, the in vitro fertilization rate of metaphase II oocytes from ATD-treated animals (9%) was reduced (P < 0.05) relative to that in controls (25%). While basal progesterone production by luteinizing granulosa cells in vitro was similar between groups, the addition of hCG in vitro enhanced progesterone secretion by cells from ATD-treated animals (3.1 +/- 0.3-fold over basal) to a greater extent (P = 0.05) than in controls (1.5 +/- 0.3-fold). Progesterone receptor was detected by immunocytochemistry in nuclei of luteinizing granulosa cells from ATD-treated animals as well as controls. Serum progesterone profiles and the length of the luteal phase were similar between groups. Thus, acute elevation of serum androgen/estrogen ratios in vivo during follicular maturation was detrimental to the gametogenic functions of the primate follicle, but did not alter follicular growth, events of early luteinization, or subsequent luteal function.(ABSTRACT TRUNCATED AT 400 WORDS)

Sperm abnormalities in retinitis pigmentosa.

PURPOSE: To determine the fatty acid composition of erythrocytes and sperm, along with the functional characteristics of sperm, in patients with retinitis pigmentosa. Sperm and retinal cells share important homologies. Both are rich in the highly polyunsaturated fatty acid, docosahexaenoic acid (DHA, 22:6[n-3]), and both contain a structural component called the axoneme. Low concentrations of DHA in the retina of monkeys are known to cause visual impairment. Because blood levels of DHA in retinitis pigmentosa patients are less than normal, reduced DHA in the retina might contribute to the visual impairment characteristic of this disease. This study was conducted on the hypothesis that the sperm of retinitis pigmentosa patients might be abnormal and that these abnormalities might infer similar lipid and structural abnormalities of the retina. METHODS: The lipid composition of erythrocytes and sperm (fatty acids and sterols) and sperm function were analyzed in 26 patients with retinitis pigmentosa and in 8 healthy men. RESULTS: The sperm of patients with retinitis pigmentosa had a much lower DHA concentration, a lower desmosterol-to-cholesterol ratio, reduced motility, abnormal structure, and lower sperm counts compared with that in normal subjects. Usher's II patients exhibited the most pronounced reductions of DHA in sperm. Sperm DHA concentration was positively correlated to sperm motility, to sperm count, and to the desmosterol-to-cholesterol ratio. Lower erythrocyte DHA was also observed in retinitis pigmentosa patients. CONCLUSIONS: These results indicate that the sperm of patients with retinitis pigmentosa, particularly those with Usher's II, have an abnormal lipid composition that is associated with reduced motility. The possibility exists that these patients might have similar abnormalities in the DHA biochemistry of the retina. Sperm biochemistry and function may be a marker for this disease. A clinical trial of DHA in retinitis pigmentosa is suggested for future study.

Antibodies to sperm-specific human FA-1 inhibit in vitro fertilization in rhesus monkeys: development of a simian model for testing of anti-FA-1 contraceptive vaccine.

The feasibility of using the rhesus monkey as a non-human primate model for testing the efficacy of a contraceptive vaccine based on FA-1 antigen was evaluated. Affinity-purified anti-FA-1 polyclonal antibodies (Fab' fragments) and anti-FA-1 monoclonal antibody were used as probes in these studies. Anti-FA-1 antibodies (polyclonal Fab' as well as monoclonal IgG) predominantly reacted with the postacrosomal, mid-piece and tail regions of rhesus monkey sperm, as with human sperm, by an indirect immunofluorescence technique (IFT). These antibodies also specifically recognized a single protein band of 51 +/- 2 kDa, corresponding to the dimeric form of FA-1 antigen, on a Western blot of lithium diiodosalicylate (LIS)-solubilized monkey sperm. Anti-FA-1 antibodies, when present in the insemination mixture, inhibited the in vitro fertilization (IVF) of monkey oocytes. These results indicate that FA-1 antigen in rhesus monkey sperm is similar in subcellular localization, molecular identity and function to that in human sperm, and that the rhesus monkey represents a permissible non-human primate model in which the efficacy of a contraceptive vaccine based on FA-1 antigen can be tested.

Protein composition of human uterine fluid.

Polyacrylamide gel electrophoresis of uterine washings and the corresponding serum from 35 patients revealed the presence of at least four characteristic uterine proteins and large amounts of the serum proteins, albumin and transferrin. The appearance of these characteristic proteins was studied during specific phases of the menstrual cycle. At lease one of the four uterine proteins was observed in postovulatory samples only, with a maximum frequency of occurrence during the midsecretory phase. In contrast, one of these proteins predominated in preovulatory uterine washings while another appeared randomly throughout the menstrual cycle. Substantial variations in protein patterns were observed in patients sampled during different menstrual cycles. The presence of the major antigenic components of serum in uterine washings was confirmed; however, immunochemical demonstration of the existence of uterine-specific antigens was unsuccessful.

Penetration of the zona-free hamster egg by human sperm.

Human sperm become capable of penetrating zona-free hamster eggs after preincubation in an appropriate culture medium. This observation has led to the development of an assay for characterizing the fertilizing capacity of human spermatozoa. In the present study, the incorporation of sperm by zona-free hamster eggs was quantitated, and several parmeters that contribute to penetration were evaluated. The importance of the motile sperm concentration was established; no penetration was seen at concentrations lower than 6 x 10(5) motile cells/ml, whereas above this level the mean number of incorporated sperm per egg was linearly related to concentration. Freeze-thawed sperm, although capable of penetrating zona-free hamster eggs, did so with lesser frequency than did fresh sperm at equal concentrations of motile sperm. Kinetic experiments indicated that eggs were maximally penetrated after 5 hours of exposure to capacitated sperm and that the cessation in sperm incorporation seen at this time resulted from egg-related changes that occurred during aging in vitro. A protocol for evaluating "fertilizing capacity" of human sperm samples was outlined incorporating the findings from the present study. Using these conditions, reproducible penetration levels were obtained when several ejaculates obtained from the same donor over a 3-month interval were tested at similar motile sperm concentrations.

Zona-free hamster eggs and human sperm penetration capacity: a comparative study of proven fertile donors and infertility patients.

An in vitro penetration assay utilizing human sperm and zona-free hamster eggs was employed to evaluate human sperm fertilizing capacity for 36 patients from the infertility clinic and 9 donors of proven fertility. The infertility patients were grouped according to the normality of their semen analyses. Test results were different for the three groups: a mean penetration level +/- standard deviation of 81% +/- 26% was obtained for proven fertile donors, while a value of 14% +/- 17% was observed for infertile couples with an abnormal semen analysis. A mean +/- standard deviation penetration level of 48% +/- 33% was associated with infertile couples in which the semen analysis was normal. These groups were statistically different (P less than 0.02) when inseminations were conducted at equivalent concentrations of motile sperm. No correlation was obvious between penetration test results and any of the parameters of the semen analysis; however, penetration test results did not correlate positively with the survival index (sperm survival at the end of insemination). These results are discussed in relation to further clinical application of the test.

A practical, objective approach to the evaluation of sperm and cervical mucus in humans.

Human sperm fertility potential, expressed as a quality index (QI), was evaluated objectively from considerations of sperm velocity, percentage of motile forms, sperm density, and ejaculate volume. Turbidimetry was applied in the quantitation of sperm velocity. High QIs (700) were characteristic of semen samples that were capable of penetrating cervical mucus in capillary tube penetration tests, while low QIs (80) were associated with specimens that did not penetrate mucus. The rate of decline in QI as a function of time postejaculation was determined for samples stored at 37 degrees C and at ambient temperature, providing correction factors for the comparative evaluation of semen samples analyzed at different times. The penetrability of cervical mucus was determined by capillary tube penetration testing. Subsequent chemical analysis of sperm-penetrable and impenetrable samples indicated that the concentrations of mucus nondialyzable solids (NDS), mucins, and soluble proteins were significantly higher in impenetrable specimens. These characteristics were also observed in impenetrable mucus obtained from patients with cervical factor infertility. These studies indicate that it is now feasible to employ mucus [NDS] in the diagnosis of cervical factor infertility and turbidimetry in the evaluation of human sperm quality.

Successful hysteroscopic cannulation and tubal transfer of cryopreserved embryos.

Clinical trials comparing nonsurgical transfer techniques with laparoscopic directed methods are needed to determine the most successful and cost-effective approach for gamete and ET. We report the successful nonsurgical transfer of frozen-thawed embryos into the fallopian tube after our initial attempt resulting in an ongoing IUP. The process is relatively simple, safe, and avoids the risks of general anesthesia. Unlike US-guided tubal cannulation, this technique offers direct, clear visualization of the tubal ostium, assuring the desired catheter placement. As well, hysteroscopic guidance allows an accurate estimate of the depth of catheter insertion within the fallopian tube, which may be a critical factor in successful tubal deposition of gametes or embryos. Whether prolonged carbon dioxide exposure of the tubal microenvironment and/or direct endometrial trauma limits the efficacy of this technique remains to be determined. Finally, outpatient hysteroscopic directed tubal cannulation holds promise as a methodological technique that ultimately assists in the examination of the best site for embryo deposition (tubal versus uterine) and/or method of delivery (nonsurgical versus surgical).

Turbidimetric analysis of human sperm motility.

A turbidimetric method has been developed for determining rapidly the fraction of sperm in human ejaculates which show the most vigorous motility. The method is based on the fact that sperm cells so endowed will be the first to swim upward into clear medium from a concentrated cell suspension at the bottom of an optical cuvette. This results in a time-dependent increase in turbidity in the medium which is recorded spectrophotometrically as an increase in absorbance. The determination requires 10 minutes and yields both the fraction of rapidly moving sperm, FRM, and their average velocity, VRM. Examination of 25 samples yielded FRM values of 10% or lower, whereas values for VRM averaged about 100 microns/second. These vigorously motile cells may be the best candidates for fertilization, and samples with a high fraction of such cells should have high fertilizing capacity. It is suggested that this simple turbidimetric test be used in evaluation of human semen as a possible indicator of fertilizing capacity.

Undetected ovulation in in vitro fertilization-embryo transfer patients.

A retrospective evaluation was done of 102 consecutive in in vitro fertilization-embryo transfer (IVF-ET) treatment cycles that culminated in surgical intervention for oocyte pickup. In 35% of these patients, a disparity was noted in the number of mature follicles present on the day of human chorionic gonadotropin administration, compared with the day of surgery. This suggests the occurrence of undetected ovulation. An endogenous luteinizing hormone (LH) surge was detected in 14 of these patients. Another cohort showed evidence of early luteinization without a detected endogenous LH surge. Finally, a group without early luteinization was defined. Possible explanations for these outcomes and the implications for success of IVF-ET are discussed.