Meiotic spindle location and identification and its effect on embryonic cleavage plane and early development.

BACKGROUND: To examine the relationship between the meiotic spindle, the first cleavage plane and any resulting influence on embryonic development parameters. METHODS: Sibling oocytes (n = 246) were allocated to either a control [polar body (PB)-aligned] or a treatment (spindle-aligned) microinjection group by use of a random numbers table. Spindles were identified by PolScope((R)) and the early embryo development parameters, and angle of first cleavage plane in relation to a defined animal-vegetal pole were analysed. RESULTS: Most oocytes (92.7%) had a visible spindle at the time of microinjection; however, 62.6% of first PBs (1PBs) were not located above the spindle (average deviation 37.3 +/- 33.2 degrees; range 0-176.6), with 6.9% of 1PBs in the opposite hemisphere to the spindle. The second PBs (2PBs) can also have an unpredictable deviation from the position of the meiotic spindle (12.5 +/- 16.7 degrees; range 0-91.8). This increased when the 1PB was above the spindle, forming a physical barrier to extrusion (average 24.7 +/- 16.1 degrees; range 7.9-91.8). Embryos developing from the spindle-aligned microinjection group had significantly more blastomeres per embryo (P = 0.044), a higher morphology score per embryo (P = 0.008) and a significantly higher average embryo score parameter (P = 0.003), with more embryos developing without any detectable fragmentation (P < 0.05) than the PB-aligned control group. Non-fragmented embryos undergo meridional cleavage, with a small angle between the spindle location and first cleavage plane (16.4 +/- 14.0 degrees ) compared with embryos with some degree of fragmentation (P = 0.002). This angle increased with the degree of fragmentation, with worst quality embryos having a spindle:cleavage angle of 45.1 +/- 17.7 degrees. CONCLUSIONS: The 1PB and, to a lesser degree, the 2PB can be unreliable predictors of the exact meiotic spindle location in human oocytes. Embryos from spindle-aligned oocytes have an increase in all measured development parameters over control siblings. When the animal pole is defined as the meiotic spindle location, non-fragmented embryos tend to develop from a meridional cleavage; with the most fragmented embryos developing from a more equatorial initial cleavage plane. This study proposes that the spindle accurately marks the animal pole in human oocytes, and provides evidence linking the meiotic spindle location to the first cleavage plane and resulting early embryo development parameters in human embryos.

Follicular flushing at oocyte retrieval: a reappraisal.

A survey of Australian assisted reproduction technique (ART) units revealed many (> 50%) use flushing of the follicle in addition to direct aspiration of the fluid during oocyte retrieval. The rationale is that flushing offers an advantage to the patient, with a larger number of oocytes being collected and thus a higher potential for pregnancy Following a complication in a patient, the medical staff determined that flushing might have been implicated. While this was later shown to be unfounded, the unit changed the method of oocyte collection, thus providing an opportunity historically to evaluate the differences between aspiration of follicular fluid alone or with additional flushing of each follicle. Thus the aim of this analysis was to test the hypothesis that aspiration alone does not effect the outcomes of ART with respect to oocyte numbers collected, their quality and subsequent fertilisation, or ultimate pregnancy rate in a large patient group (n = 2378). During the review period (1991-1993) the manufacturer of the aspiration needles; the pump and pressure used for aspiration; the staff involved in all procedures; and the premises, equipment and media used for oocyte collection and culture remained constant. Similarly the two patient groups did not differ in their demographics and physical characteristics. There was no difference (p > 0.5) in the number of oocytes collected, the number of embryos created or the pregnancy rate for that treatment cycle. There was also no significant difference between the two groups in fertilisation rates, irrespective of the type of treatment being used in vitro fertilisation (IVF), gamete intrafallopian transfer (GIFT) or intracytoplasmic sperm injection (ICSI)). The data presented in this paper are not prospective. They use historical comparison and are confounded by minor changes in ovarian stimulation protocols, but the conclusions are still valid. These data suggest there is no difference in the outcome of ART (from oocyte collection through to pregnancy rate) whether or not aspiration of follicles is accompanied by flushing. First principles of surgery advocate the shortest possible operating time, the simplest procedure and minimum amount of tissue handling as maxims for reducing complication. Therefore, as a routine, flushing would seem superfluous in ART.

Oocyte morphology and early zygote cleavage does not appear to be related to intrafollicular concentrations of inhibin-A or -B.

PURPOSE: The aim was to investigate the relationship of intrafollicular inhibin dimers A and B with human oocyte morphology and subsequent embryo potential. METHODS: Sixty-eight oocytes were isolated from 31 women undertaking intracytoplasmic sperm injection (ICSI). Estradiol, progesterone, testosterone, luteinizing hormone (LH), follicle-stimulating hormone (FSH), prolactin, sex hormone-binding globulin, inhibin-A and inhibin-B was assayed in corresponding follicular fluid. RESULTS: The mean (+/- SD) concentration for inhibin-A was 9.7 +/- 9.8 ng/ml (range, 1.1-60.0 ng/ml) and for inhibin-B was 269.4 +/- 185.2 ng/ml (range, 33.1-811.0 ng/ml). In a correlation matrix there were no marked relationships (r < 0.556) between inhibin and steroid or gonadotropin concentrations. Similarly, when inhibin concentrations were divided according to whether the oocytes had mature or immature cumulous complexes, were viable or necrotic, were meiotically immature or mature, became fertilized or not, or had different embryo gradings after cleavage, no statistically significant difference could be seen between groupings. CONCLUSIONS: Because the range of values was large and the data often skewed, neither inhibin dimer has discriminatory power to reflect the potential of the oocyte.

A prospective, randomized, controlled, double-blind, double-dummy comparison of recombinant and urinary HCG for inducing oocyte maturation and follicular luteinization in ovarian stimulation.

A randomized, controlled, double-blind, double-dummy, phase III clinical trial was conducted in 84 women to compare the efficacy of a s.c. injection of 250 microg recombinant human chorionic gonadotrophin (rHCG; Ovidrel) to an i.m. injection of 5000 IU urinary HCG (uHCG; Profasi) in inducing folliculogenesis, resumption of oocyte meiosis and luteinization after ovulation induction with recombinant follicle stimulating hormone (Gonal-F). The study primary endpoint was comparison of the number of oocytes retrieved per patient receiving either compound. Secondary comparisons included the number of oocytes retrieved per follicles aspirated; the number of mature oocytes; normally fertilized oocytes; and cleaved embryos. There were no statistically significant differences between groups for the primary endpoint (mean +/- SD oocytes retrieved 10.8 +/- 4.5 for rHCG versus 10.3 +/- 5.1 for uHCG) or each of the secondary endpoints except for increased concentrations of progesterone 6-7 days after rHCG administration (353.2 +/- 215.1 versus 234.1 +/- 129.4 nmol/l; P < 0. 004) and for HCG during the luteal phase following rHCG (P < 0.02). There were also no significant side-effects for either drug. Since the confidence intervals for the difference of the number of oocytes retrieved between the two treatment groups were within the bounds defined by the multi-trial protocol equivalence between rHCG and uHCG could be declared.

Failure to collect oocytes in assisted reproductive technology: a retrospective.

While there is much information and discussion on pregnancy failure after assisted reproductive technologies, less emphasis is placed on the failure to collect oocytes after apparently successful ovarian stimulation. This retrospective survey reviewed 4973 treatment cycles in order to obtain information about the likelihood of this event. Overall 42 women (43 treatment cycles) failed to have oocytes collected [0.86% of treatments started and 0.92% of women given human chorionic gonadotrophin (HCG)]. However, in only six cases was this failure unexpected (0.1%) with no obvious potential clinical reason (i.e. all six cases had: HCG administered; more than two follicles >15 mm in diameter; oestradiol values >2000 pmol/l; <38 years old; normal body mass index). Indifference concerning uncommon events is fraught with peril, as although rare, the particular outcome may be devastating to the individual, both economically and psychologically. Eighteen of the 42 women did not return for on-going treatment suggesting increased contact by clinic staff may be required when oocyte retrieval is not achieved. These data suggest that the failure to collect oocytes after apparently successful ovarian stimulation is rare and random. The information has proved useful in allaying the fears of couples contemplating assisted reproductive technologies.

Factors affecting pregnancy outcome resulting from assisted reproductive technology (ART).

OBJECTIVE: A retrospective, observational study of pregnancy outcome was performed on variables maintained in an ART database to determine factors that might affect miscarriage rate in pregnancies resulting from assisted reproduction technologies (ART). METHODS: Previously infertile couples, where conception was achieved after ART, were included. Seven hundred and ninety-four consecutive clinical pregnancies, diagnosed by ultrasound documentation of the gestation sac in the first trimester were divided into 2 groups: 'miscarriage' and 'term birth'. Differences between the groups were analysed using crosstable regression analyses or t-test in second yearly cohorts. RESULTS: A statistically significant positive relationship was seen between age and spontaneous abortion rate (p = 0.008) with a major increase after the age of 38 years. There was no significant difference in the mean number of oocytes retrieved between groups (p = 0.17). While there was a significant negative correlation between maternal age and the total number of oocytes collected (p < 0.001), there was no statistical difference between those women who miscarried or delivered a live infant. No relationships were found with any other variables analysed. CONCLUSION: Maternal age is probably the most important factor in pregnancy outcome in ART. This survey could not determine any other variables as being major prognostic determinant for miscarriage once pregnancy was attained.

Variation in the determination of follicular diameter: an inter-unit pilot study using an ultrasonic phantom.

Ultrasound operators in assisted reproductive technology units in New South Wales, Australia cooperated in a study that was conducted to assess their ability to measure fixed objects embedded in an ultrasound phantom. The results have demonstrated a large variation ranging between 10 and 25% coefficient of variation on distances between 10 and 32 mm. As ultrasonic imaging is the only component of assisted reproductive technology not currently controlled by an external quality assurance scheme, the authors suggest there may be a need to establish a programme.

Is it Sertoli cell only syndrome? A case study and clinical caution.

The use of the terminology Sertoli Cell Only Syndrome should be confined to its classic definition (ie; total absence of germinal cells) when reporting the results of a testicular biopsy, since with intracytoplasmic sperm microinjection (ICSI) the presence of any mature sperm in a histological section may mean that the patient has a chance of using his own gametes for attempted pregnancy.

Transfer of gametes into the fallopian tubes--is choice of side important?

The aim of this study was to define pregnancy rates for gamete intra-Fallopian transfer (GIFT) with respect to the ovary from which most oocytes were collected (i.e. a gross index of ovarian stimulation) and the Fallopian tube (ipsilateral or contralateral) into which gametes were replaced. The only inclusion criterion was the ability to transfer gametes into the Fallopian tubes at GIFT. No other factors that could influence outcome were considered. In this retrospective review of the clinic's database, no relationship was found between pregnancy rate and placement of gametes into the Fallopian tube coincident with the ovary from which most oocytes were collected. Thus when performing unilateral tubal transfer at GIFT gametes may be returned to the side most convenient to the operating surgeon without fear of compromising pregnancy potential.

Temperature change in cryo-containers during short exposure to ambient temperatures.

Times have been defined for the handling of 0.25 ml embryo cryostraws and semen, in either 0.5 ml cryostraws or 1.0 ml cryovials containing 0.5 ml material, before potentially detrimental changes in temperature take place. When handling cryovials the time lag is relatively long, with 78.8 +/- 2.6 s being available to manipulate the vials before -80 degrees C is reached and 335.4 +/- 3. 8 s until the eutectic point (approximately -7 degrees C) is reached. However the situation with cryostraws is less tolerant. Both 0.25 and 0.5 ml versions reach temperatures >-80 degrees C within 40 s, and the eutectic point is reached in 79.0 +/- 2.0 s in 0.25 ml cryostraws. These time/temperature data have proved useful in educating new technicians, as well as clinicians and nurses who may also handle frozen human material, in the need to minimize the ambient temperature exposure time of stored specimens so as to maintain optimal post-thaw viability.

Hyperspermia: the forgotten condition?

The seminal volumes of 4223 men with known periods of abstinence of ejaculation, complete collections and with no dysfunction known to affect production of accessory gland secretions have been examined to define a minimum value for hyperspermia. The 95th percentile of the skewed data distribution was 6.3 ml and of the 229 men with values equal to or greater than this, 113 (49.3%) had sperm concentrations below the World Health Organization accepted minimum 'normal' value of 20 x 10(6)/ml. Basic seminal parameters should not be forgotten when assessing infertile men.

The effect of maternal serum on phase-change temperature in human embryo cryopreservation.

The effect of addition of maternal serum to cryomedium on the phase-change temperature for embryo cryopreservation has been investigated. No effect on the eutectic point (i.e., earliest seeding temperature) was found within patients because of serum concentration or the time in the ovarian cycle when the serum was collected. However, the optimum seeding temperature did vary between patients, which suggests that self-seeding devices for embryo cryopreservation may be practically beneficial.

Positive chlamydial serology and its effect on factors influencing outcome of IVF treatment.

A retrospective study of 98 IVF treatment cycles from 49 couples has assessed the relationship between presence of antibodies to Chlamydia trachomatis and success with respect to fertilization, cleavage and pregnancy rate, both at nidation and term. No effect of prior infection on IVF outcome was noted.

Chlamydia trachomatis: the prevalence, trend and importance in initial infertility management.

One thousand and five women, and 354 of their partners, attending an infertility clinic have been assessed for the presence of Chlamydia trachomatis antibodies in their serum. The overall prevalence in women was 12.4% (125/1,005) but appeared to have increased over a 2-year period. A similar trend was not apparent for men and the prevalence was only 4.2% (15/354). Of the 125 seropositive women 81 had their pelvis examined for the first time by laparoscopy. Fifty seven had tubal disease of varying severity, but only 16 had had an ectopic pregnancy or admitted to a previous history of pelvic inflammatory disease or nonspecific urethritis. These findings suggest that laboratory testing for chlamydial antibodies should be routine in the initial investigation of an infertile couple and that early laparoscopy is indicated in seropositive women.

Cervical carcinoma: a comparison of four potential biochemical tumor markers.

A longitudinal study of circulating immune complexes (CIC), cancer antigen 125 (CA125), carcinoembryonic antigen (CEA) and a sub-fraction of the TA-4 squamous cell carcinoma tumor-associated antigen (SCC) has been undertaken in 38 patients with cervical carcinoma. Pre- and post-treatment values have been compared with those obtained in well-defined clinical remission and relapse phases of their disease. Each tumor marker was assessed in terms of "lead time" before clinically obvious recurrent disease became evident. The data from the four subjects with adenocarcinoma of the cervix gave equivocal results and no firm conclusions could be drawn. However, for the 34 patients with squamous cell carcinoma the medium value (data was skewed) for SCC was elevated above normal in the presenting pretreatment sera (4.5 ng/ml) and significantly fell to 2.5 ng/ml post-treatment (P less than 0.01). A similar pattern was not apparent for CIC, CEA, or CA125 data. When results were examined for an individual patient, of those with recurrent squamous cell lesions who died, 12/24 demonstrated elevated, and rising SCC values before clinical evidence of the disease and a further 6 (25%) at the time recurrence was clinically evident. This information gave lead times of between 2 and 52 months (median 13 months) for 75% of patients. Only 1 subject had values which remained in the normal range (less than 2 ng/ml) even though their disease was progressive. Similarly of the subjects still in clinical remission 8/9 had values within the normal range. The data for CIC, CA125, and CEA were not individually useful as a marker. Furthermore, combining the data from all analytes to give a panel of potential markers did not improve the prognosis already evident with SCC alone. It has therefore been concluded that SCC is a useful biochemical marker of the progression of squamous cell carcinoma of the cervix.

Chorioamnionitis: how useful is the determination of C-reactive protein?

This study was designed to derive the predictive value of C-reactive protein (CRP) in peripheral venous serum of patients admitted to hospital with suspected premature rupture of the membranes (PROM). CRP was assayed by each of 4 separate methods and the results have been compared for accuracy and practical value with respect to clinical outcome and the histopathology of the placenta. Of the 4 techniques used only the latex test had characteristics suitable for a diagnostic screen. While the results were only semiquantitative, when comparisons were made to other techniques no significant change in clinical diagnosis would have been made. The results have confirmed that chorioamnionitis and preterm labour are often associated, but in some instances the extent of inflammatory infiltration was greater than might have been expected from the short time interval between documented membrane rupture and delivery. Thus it may be speculated that some cases of PROM are secondary to, rather than causative of, infection. Finally it is suggested that a controlled therapeutic trial of active intervention in those cases of PROM with elevated CRP in the absence of other clinical parameters suggestive of intrauterine infection should be undertaken.