Optimising vitrification of human oocytes using multiple cryoprotectants and morphological and functional assessment.

Oocyte vitrification is a clinical practice that allows preservation of fertility potential in women. Vitrification involves quick cooling using high concentrations of cryoprotectants to minimise freezing injuries. However, high concentrations of cryoprotectants have detrimental effects on oocyte quality and eventually the offspring. In addition, current assessment of oocyte quality after vitrification is commonly based only on the morphological appearance of the oocyte, raising concerns regarding its efficiency. Using both morphological and functional assessments, the present study investigated whether combinations of cryoprotectants at lower individual concentrations result in better cryosurvival rates than single cryoprotectants at higher concentrations. Surplus oocytes from IVF patients were vitrified within 24h after retrieval using the Cryotop method with several cryoprotectants, either individually or in combination. The morphological and functional quality of the vitrified oocytes was investigated using light microscopy and computer-based quantification of mitochondrial integrity, respectively. Oocyte quality was significantly higher using a combination of cryoprotectants than vitrification with individual cryoprotectants. In addition, the quality of vitrified oocyte varied depending on the cryoprotectants and type of combination used. The results of the present study indicate that observations based purely on the morphological appearance of the oocyte to assess the cryosurvival rate are insufficient and sometimes misleading. The outcome will have a significant implication in the area of human oocyte cryopreservation as an important approach for fertility preservation.

The management of oligospermic infertility by in vitro fertilization.

The fertilization rates of mature preovulatory oocytes aspirated from 156 women treated by in vitro fertilization were analyzed as a function of spermatozoal density and motility and the findings were correlated with the category of infertility (chronic oligospermia, tubal disease, endometriosis, serum antibodies to spermatozoa in the female, and unexplained infertility). Overall reduced fertilization rates were found in all cases if the semen sample presented on the day of fertilization demonstrated less than or equal to 5 million motile spermatozoa per milliliter, less than 40% motile forms, or the combined findings of less than 20 million per ml and less than or equal to 60% motile forms. Where the husband was known to have chronic oligospermia, reduced fertilization was found if the semen on the day of fertilization contained less than 20 million spermatozoa per ml and less than 12 million motile spermatozoa per milliliter. For cases of unexplained infertility, a poor fertilization rate was noted if the semen demonstrated less than 60% progressively motile forms regardless of the overall spermatozoal density, implying that a proportion of unexplained infertility is due to a disorder of spermatozoa reflected by reduced motility. Pregnancies were achieved in 5 of 26 cases with chronic oligospermia, including 2 where oligospermia was very pronounced (less than or equal to 5 million motile spermatozoa per milliliter).

Combined pregnancy after gonadotropin therapy.

Three cases of combined pregnancy are described after gonadotropin therapy; two cases after human pituitary gonadotropin and one after human menopausal gonadotropin administration. In each case the intrauterine gestation was a multiple pregnancy. After salpingectomy, two of the women have proceeded to the delivery of healthy infants; the third woman aborted. In each case the gonadotropin stimulation regimen was ceased at the appropriate stage when the estriol excretion was between 60 and 125 micrograms/day, but the subsequent rate of rise of estriol was 2.3- to 3.2-fold during the coasting phase before the human chorionic gonadotropin trigger when the estriol excretion rate was 140 to 350 micrograms/day.

Failure of human oocyte release at ovulation.

Among 150 patients admitted for ovum aspiration, in vitro fertilization, and embryo transfer in Perth, Western Australia, 14 were found to have had at least one ovulated follicle at the time of laparoscopy. Based upon ultrasound estimation of follicle diameter 24 hours previously, ovulation occurred in 6 of 22 follicles less than 1.7 cm in mean diameter and in 16 of 19 follicles greater than 1.7 cm in mean diameter. When the ruptured follicles were flushed with heparinized fertilization medium, oocytes were collected in 13 of the 22 dispersed follicles. Twelve oocytes developed pronuclei 16 hours after the addition of spermatozoa, and 11 cleaved to the 4-cell stage within 44 hours of insemination. Oocytes were recovered from 6 of 11 patients stimulated with clomiphene alone and from all 3 patients stimulated with clomiphene supplemented with human menopausal gonadotropin. These observations suggest that oocyte release and follicle rupture are not necessarily synonymous events and that the incidence of retained oocytes in ovulated follicles following stimulation with clomiphene or clomiphene plus human menopausal gonadotropin could be on the order of 60%.

Prolonged follicle stimulation decreases pregnancy rates after in vitro fertilization.

Ovarian stimulation with a GnRH-a and hMG for IVF treatment offers the potential to extend the period of follicle recruitment and growth free of the restriction imposed by a spontaneous midcycle LH surge. A randomized trial investigating the impact of extending follicle growth by 1 day resulted in a significantly reduced PR, despite an increase in the number of larger follicles (greater than or equal to 17 mm) at the time of hCG. The reason for a reduced PR was not related to the number of ova recovered or embryo quality. This suggests that follicular aging or the duration of estrogen rise may be important.

An evaluation of peripheral blood platelet enumeration as a monitor of fertilization and early pregnancy.

This paper reports our data that confirm the existence of early pregnancy-associated thrombocytopenia (EPAT) in the mouse and illustrate that the phenomenon is independent of age, parity, and strain differences. This paper also provides evidence that the EPAT phenomenon is induced by a soluble factor (EPAT-factor) released by the fertilized ovum. EPAT-factor was produced in vitro by mouse embryos from the 1-cell stage to the expanded blastocyst stage. The human study involved a "blind" analysis of serum samples, collected from in vitro fertilization-treated patients, for the presence of thrombocytopenic activity. Results suggest that measurement of this thrombocytopenic activity might be useful as an index of embryo viability and might be clinically applicable for the monitoring of implantation success in in vitro fertilization programs.

Monozygotic twins from in vitro fertilization.

A case of identical twins following in vitro fertilization and embryo transfer ( IVF -ET) is described. Two embryos were transferred, but it is apparent that only one implanted and subsequently divided in the early implantation phase to produce identical male twins within a monochorionic , diamniotic placental and membrane configuration. Additional marker studies provide an overall probability of less than 0.001 for dizygosity . There is unlikely to be any relationship between this event and the technique of IVF -ET.

Antispermatozoal antibodies in human follicular fluid.

The possibility of antispermatozoal antibodies in women having significant effects in the higher regions of the female reproductive tract has been investigated. Follicular fluids (FF) and sera taken at the time of oocyte recovery from women undergoing in vitro fertilisation and embryo transfer (IVF-ET) were tested for the presence of antispermatozoal antibodies, and the concentrations of IgM, IgG, IgA, and complement C3 were determined. The concentrations of immunoglobulins and C3 in FF were consistent with transudation from serum inversely proportional to molecular weight. Titres of agglutinating and immobilizing antibodies in FF were usually one or two dilution steps below those of serum except where immobilizing activity was associated with IgM. IgG:IgA ratios were lower in FF from women with antispermatozoal antibodies, suggesting local production or enhanced transudation of IgA; however, a secretory component could not be detected in any of the follicular fluids in this study. Two women with antispermatozoal antibodies and infertility in excess of 5 years had successful IVF-ET and have delivered healthy infants.

Mechanistic studies of early pregnancy associated thrombocytopenia (EPAT) in the mouse.

The preimplantation period of uterine pregnancy is associated with the transient (first 4 days of gestation) expression of a state of early pregnancy-associated thrombocytopenia (EPAT), a phenomenon shown to be mediated by the embryo-derived EPAT factor, which presumably causes platelet activation and subsequent removal. We previously investigated the time course of production of EPAT factor in mouse embryo culture medium and found a correlation between the production of this factor and the in vivo platelet alterations in pregnant mice. The present paper supports the postulation that the EPAT factor and PAF-acether (a phospholipid platelet-activating factor) are related by providing data showing that PAF-acether may be responsible for the thrombocytopenia. Finally, data are presented to suggest that platelet activation, though not affecting the rate of ovulation, is important for successful ongoing pregnancy. Results suggest that the EPAT factor, produced by the fertilized egg, might act to signal uterine decidualization and/or modulate maternal immunological rejection of the implanting conceptus.

Custom versus standardized patient satisfaction surveys: a Pacific Telesis case study.

With the increased interest in and use of patient satisfaction surveys has come the search for the ideal survey that could be used as a definitive tool to measure satisfaction. This article overviews the advantages and disadvantages of custom versus standardized patient satisfaction surveys. A disenrollment survey conducted by Pacific Telesis in 1994 and 1995 serves as a case study of a useful custom survey.

Potentially important variables identified by transvaginal ultrasound-guided embryo transfer.

Transvaginal ultrasound-guided embryo transfer was performed on 121 consecutive patients. Observation was made of guiding cannula and transfer catheter placement in relation to the endometrial surface and uterine fundus during embryo transfer. The position and movement of a transfer-associated air bubble and the impact of subendometrial myometrial contraction leading to endometrial movement was observed. Results indicate that tactile assessment of embryo transfer catheter placement is unreliable: in 17.4% of transfers the outer guiding catheter inadvertently abutted the fundal endometrium. The outer guiding cannula indented the endometrium in 24.8% and the transfer catheter embedded in the endometrium in 33.1%. Unavoidable sub-endometrial transfers occurred in 22.3% of transfers. Ultrasound-guided transfer avoided accidental tubal transfer in 7.4% of transfers. Transfer catheter withdrawal did not significantly affect embryo transfer-associated air bubble position. Endometrial movement due to sub-endometrial myometrial contraction was obvious in 36.4% of cases, with active motion of the transfer-associated air bubble occurring in 28.1%. Pregnancies occurred in 45.5% of transfers with endometrial movement compared to 15.6% (P < 0.001) without.

Ultrasound tracking of the movement of embryo-associated air bubbles on standing after transfer.

The aim of this study was to investigate whether standing upright shortly after embryo transfer has any potential to affect the position of embryos transferred to the uterine cavity during treatment with in-vitro fertilization (IVF). This was assessed by ultrasound-guided tracking of embryo-associated air within the uterine cavity. A prospective study of 93 patients undergoing 101 consecutive embryo transfers in an IVF programme was carried out. Transvaginal ultrasound guided embryo transfer was performed with a second ultrasound in standing position immediately after transfer, allowing the movement of embryo-associated air to be assessed. No movement occurred in 94.1% (95/101) of transfers, movement of <1 cm in 4.0% (4/101) of transfers and movement of 1-5 cm in 2.0% (2/101) transfers. No movement of embryo-associated air out of the uterine cavity, either into the cervix or the intramural portion of the Fallopian tube, was seen. Standing shortly after embryo transfer does not play a significant role in the final position of embryo-associated air and is unlikely to be a factor in determining the position of embryos transferred to the uterine cavity during treatment with IVF.

The fluid dynamics of injection: variables as they relate to transvaginal gamete intra-fallopian transfer and tubal embryo transfer.

Transvaginal gamete intra-Fallopian transfer (GIFT) and tubal embryo transfer have not, in most cases, produced pregnancy rates as high as the equivalent transabdominal methods. We postulated that two parameters, i.e. wide dispersion of the gametes and peritoneal spill of gametes or embryos, are of prime importance in explaining this difference. We designed this study to analyse the effect of varying the rate of injection (microliter/min) of radio-opaque contrast media and the distance the transfer catheter is placed into the tubal isthmus upon these two parameters. Selective salpingography was performed on 30 patients who were allocated to one of two experiments. In the first experiment (flow rate), 20 patients were divided into four groups so that we could analyse four sets of 10 Fallopian tubes. Each group was subjected to injection of contrast at different flow rates: 100, 50, 25 or 12.5 microliters/min. Peritoneal spill occurred from 4, 2, 0 and 0 tubes and wide dispersion (> 4 cm) of contrast occurred in 6, 5, 1 and 0 tubes at the respective injection rates of 100, 50, 25 and 12.5 microliters/min. At 12.5 microliters/min contrast was localized to < 2 cm of tubal ampulla in all cases. In the second experiment (catheter placement), the remaining 10 patients were divided to allow analysis of two groups of 10 Fallopian tubes. The transfer catheter was placed either 1.5 cm or 5 cm into the tube and the radio-opaque contrast was injected at 12.5 microliters/min. No difference in the degree of dispersion of contrast along the Fallopian tube was observed. No backflow of contrast into the uterine cavity was observed in any patient following removal of the catheter.(ABSTRACT TRUNCATED AT 250 WORDS)

The relationship between motility and the FITC-BSA binding properties of mouse epididymal spermatozoa.

Mouse epididymal spermatozoa exposed to fluorescein isothiocyanate-conjugated bovine serum albumin (FITC-BSA) immediately following dilution or after a 2-hour incubation period under fertilization conditions, were assessed by fluorescence microscopy for albumin adsorption. Motile spermatozoa exhibited light fluorescence in the midpiece and tail but not in the head. In contrast the majority of nonmotile spermatozoa displayed a strong and characteristic fluorescence in the post acrosomal region of the sperm head as well as the midpiece. Spermatozoa immobilised by short-term heat stress exhibited fluorescence in the post acrosomal region and midpiece as before but also in the acrosomal cap. The equatorial region failed to fluoresce. The significance of these observations on the involvement of albumin in capacitation is discussed.

The limitations of in vitro fertilization from males with severe oligospermia and abnormal sperm morphology.

Thirty-one patients whose infertility was attributed to oligospermia were included for treatment by in vitro fertilization and embryo transfer. Three subgroups were defined: severe oligospermia (less than or equal to 5 million motile sperm/ml), moderate oligospermia (6 to less than 12 million motile sperm/ml), and abnormal sperm morphology (greater than 60% atypical). The fertilization rates were compared to those of a normospermic group managed concurrently. A modified overlay technique of sperm preparation is described for oligospermic samples so that the number of motile spermatozoa inseminated into each tube or culture dish containing a mature preovulatory oocyte was similar in each category, within the range 0.5 to 2 X 10(5)/ml. Significantly fewer oocytes were fertilized in the severe oligospermic group (P less than 0.001), suggesting a reduced capacity for fertilization by spermatozoa from severely oligospermic males. The fertilization rate of oocytes was normal in the moderate oligospermic group and those with abnormal morphology, although in the latter there was a significant delay noted in reaching the pronuclear stage (P less than 0.001), and the embryos were at a less advanced stage of cleavage at the time of transfer (0.001 less than P less than 0.01). Pregnancies were achieved in both the severe and the moderate oligospermic groups, with healthy infants delivered from each.

Reduced in-vitro fertilization of human oocytes from patients with raised basal luteinizing hormone levels during the follicular phase.

A series of 62 women were managed in the University of Western Australia/PIVET Laboratory in-vitro fertilization programme. In 60 of them follicle growth was stimulated with clomiphene citrate with or without additional human menopausal gonadotrophin (hMG) and in two with hMG alone. Follicles were aspirated at laparoscopy following an hCG trigger injection and occasionally following a spontaneous luteinizing hormone (LH) surge. Oocytes were inseminated with 0.5 X 10(5)-10(5) sperm/ml 3-6 h later. A significant reduction (P less than 0.001) in the fertilization rate of mature oocytes was observed in those patients whose basal serum LH values were greater than 1 SD above the mean. Fifty-nine women subsequently had embryo transfer and of 10 clinical pregnancies, none occurred in those with elevated LH values. Reduced fertilization may be a reflection of premature oocyte maturation or ageing. This may have clinical implications in the management of some patients with unexplained infertility.

Fertilization of pronase-treated mouse ova in vitro.

In order to obtain consistently a large number of zona-free mouse ova for studies of sperm-egg interactions, a study was made of the relative effectiveness of removing the zona pellucida from ova by mechanical or enzymatic treatments. Ova exposed to pronase before mechanical removal of the zona pellucida in medium devoid of pronase had similar fertilization rates in vitro compared with ova mechanically denuded in the absence of pronase. Ova with pronase-weakened zonae were easier to denude and survived the mechanical manipulations better than the ova denuded by vigorous aspiration in narrow-bore pipettes. However, exposure to pronase did significantly lower the incidence of polyspermy in the naked ova, indicating that some of the enzyme may have diffused across the perivitelline space and damaged sperm-binding sites on the vitelline plasma membrane. The enzyme treatment also reduced the fertilization rate of zona-intact ova.

Effect of polyamines on fertilization of mouse ova in vitro.

After a 1-hour preincubation of epididymal mouse spermatozoa at a concentration of 2 X 10(6)/ml in 0.23 mM spermine, the proportion of F1(C57BL X CBA) mouse ova fertilized after 1 and 2 hours was significantly greater than with untreated spermatozoa. Spermine also significantly increased the proportion of ova fertilized at the sub optimal sperm concentration of 2 X 10(5)/ml. The stimulatory effect was lost when the protein source in the fertilization medium was changed from human serum albumin V (HSA) to HSA crystalline. This provides indirect evidence that albumin is directly involved in the capacitation process and that the crystalline is more potent than the fraction V preparation. At equimolar concentrations, spermidine was partially and putrescine was totally inhibitory to fertilization. Mechanisms whereby spermine may affect metabolic activity or sperm-zona binding are discussed. It is suggested spermine may also be present in ovulatory fluid and therefore could potentially be involved in fertilization in vivo.

Differential impact on pregnancy rate of selective salpingography, tubal catheterization and wire-guide recanalization in the treatment of proximal fallopian tube obstruction.

A total of 66 patients with proximal Fallopian tube (113 tubes) obstruction, as diagnosed by both laparoscopy and hysterosalpingogram, were each subjected to a transcervical recanalization procedure sequentially using selective salpingography followed, if necessary, by tubal catheterization with a soft Teflon 2-French catheter and finally, if needed, wire-guide cannulation. Each procedure were terminated once patency had been achieved without recourse to the next technique. Bilateral obstruction was present in 47 patients and unilateral in 19 patients. Patency was achieved in 39 (34.5%) Fallopian tubes by selective salpingography alone, in 52 (46.0%) by tubal catheterization and in 10 (8.9%) by wire guide, with 12 (10.6%) tubes remaining obstructed. Pregnancy occurred in 24 (36.4%) patients without recourse to other treatment (mean follow-up, 17 months). Where patency was achieved (59 patients), 19 out of 43 (44.1%) of those treated for bilateral obstruction and five out of 16 (31.3%) of those treated for unilateral obstruction achieved a pregnancy. Pregnancy occurred in six out of 22 patients (27.3%) where selective salpingography was used to produce tubal patency, in 17 out of 30 patients (56.7%) where tubal catheterization was used and in one out of seven (14.3%) where a wire guide was used, which was an ectopic pregnancy. The difference between the ongoing pregnancy rates following tubal catheterization (50.0%) and wire-guide cannulation (0.0%) was significant (P = 0.033). While wire-guide cannulation is the most effective method used to achieve tubal patency, these results indicate that when it is truly necessary, as opposed to electively used by clinicians, the prognosis with regard to pregnancy is poor and alternative therapy such as microsurgery or in-vitro fertilization should be considered early.

Pregnancy following fertilization of zona-free, coronal cell intact human ova: Case Report.

A pregnancy by intracytoplasmic sperm injection (ICSI) of a couple whose oocytes were recovered without a zona pellucida is reported. Previous IVF/ICSI cycles indicated all oocytes had either very thin or no zona pellucida at the time of cumulus removal prior to ICSI. As a result, many ova were very fragile and lysed upon handling. In the latest attempt, six ova were recovered. After coronal cell dissection of two ova where both lacked a zona pellucida and one lysed, it was elected to attempt to inject the remaining four ova without removal of their coronal cells. The zona pellucida was not visible through the cell layer and the position of the polar body in three ova was speculative. Two embryos possessed normal pronuclei on day 1, and displayed six blastomeres prior to transfer on day 3. During preparation for transfer, sufficient coronal cells fell away to confirm both embryos lacked a zona pellucida. Pregnancy was confirmed by ultrasound observation of a fetal heart 4 weeks later. This report documents that where the zona pellucida is absent, the coronal cells provide sufficient support to maintain blastomere interaction and embryo viability and the zona pellucida is not essential for early embryo development and pregnancy.