Artificial Reproductive Techniques Research Articles

Association Between the Rates of Multiple Maternities

We analyze the association between the rates of multiple maternities. Correlation analysis is suitable if there are at least monotonic relationships between the variables. A decreasing tendency can be observed in the rates of multiple maternities in Sweden and in its 25 counties for the period 1751-1960 and the effect of external variables can be assumed to be monotonic. After 1960, the rates of multiple maternities show marked increases, mainly caused by ovulation stimulants and the introduction of other artificial reproduction techniques. We transformed the triplet and quadruplet rates according to Hellin's law in order to obtain rates on the same scale as the twinning rate. The time trends for the multiple maternities in Sweden as a whole were quite similar. The regional differences in the twinning rates could not be satisfactorily explained by maternal age. Alternative attempts to eliminate the effect of other time-dependent factors were to study partial correlation coefficients when the time was kept fixed and the correlation coefficients based on cross-sectional regional data. Both the ordinary and the partial correlation coefficients showed strong regional variation. Cross-sectional analyses gave correlation coefficients similar to the partial correlation coefficients for Sweden as a whole. The variations in the correlation coefficients between the twinning and the triplet rates seem to be caused by other time-dependent factors and the effects of these factors show strong regional variation. After elimination of such factors, the correlation between the twinning and the triplet rates is moderate.

The neutral comet assay detects double strand DNA damage in selected and unselected human spermatozoa of normospermic donors.

The occurrence of DNA breaks in human sperm is of concern to genetic safety in artificial reproduction techniques. Here, we have explored the neutral comet assay (NCA) for evaluating the frequency of spermatozoa with double strand (ds) DNA breaks in normospermic donors. The NCA results into DNA tail formation by fibre extension and by the separation of DNA fragments. Gamma-irradiated native, lysed and lysed plus RNA and protein degraded human sperm nuclei have been used to assess sensitivity and specificity of fragment formation as an indication for ds DNA breaks. At 5 and 10 Gy gamma irradiation, the sensitivity increases in the order: native, lysed, lysed plus RNA and protein degraded. At 10 Gy, a uniform response between donors was obtained. For technical and biological reasons, the NCA underestimates the true incidence of ds DNA breaks by an unknown factor. Semen samples of six healthy normospermic donors were differentiated by swim up and by Percoll density centrifugation, followed by the NCA. In native semen, percentages of sperm nuclei with ds DNA breaks ranged from 15 to 25%. Swim up and selection for high-density sperm nuclei (high Percoll fraction) reduced the frequency of sperm with ds DNA breaks by about one third, whereas an increased frequency was found in the low Percoll fraction. In conclusion, the response to gamma irradiation of DNA fragment formation indicates the NCA to demonstrate ds DNA breaks which is in keeping with theory and experimental results from somatic cells. Ds DNA breaks are a characteristic of the sperm population of normal donors. Current sperm selection procedures reduce the fractions of sperm with ds DNA breaks, yet are not effective in eliminating these cells.

Longitudinal changes in plasma glucose values of the 75-g glucose tolerance test in triplet pregnancies.

We evaluated longitudinal changing patterns of 75 g glucose tolerance test (GTT) in triplet pregnancies. Eight triplet pregnancies were prospectively studied. All triplet pregnancies were fertilized with artificial reproductive techniques; patients showed no glucosuria or fasting hyperglycemia > 100 mg/dL before pregnancy. The 75-g GTT was performed at first, second, and third trimester, as well as at postpartum. Longitudinal changes in glucose levels at fasting, 1 hour, and 2 hours were compared by one-way repeated-measure analysis of variance (ANOVA) and Bonferroni/Dunn test ( p < 0.05). Values were expressed as mean +/- standard deviation. Each of the 3-point values of 75-g GTT decreased after 28 weeks of gestation in triplet pregnancies. During the third trimester, fasting values were significantly decreased compared with postpartum values (65.5 +/- 13.4 versus 74.6 +/- 4.0 mg/dL), and 2-hour values were significantly decreased from those of second-trimester 75-g GTT (116.3 +/- 19.5 versus 99.6 +/- 17.1 mg/dL). Longitudinal glucose values of 75-g GTT improve during third trimester in triplet pregnancies, suggesting that fetoplacental fuel drain may counterbalance maternal insulin resistance.

Pregnancy in a couple with a male partner born with severe bladder exstrophy

Bladder exstrophy is a rare congenital condition, with variable degrees of reproductive dysfunction, from mild exstrophy allowing regular intercourse and reproduction, to severe forms with significant cosmetic deformity and failure to reproduce spontaneously. This is a case report of a patient with severe congenital bladder exstrophy and failure to reproduce spontaneously due to microphallism and anejaculation, treated with testicular sperm aspiration and intracytoplasmic sperm injection (ICSI), resulting in the conception and birth of a healthy unaffected singleton male infant. This the first case report of bladder exstrophy of such a severe degree with a successful reproductive outcome. It illustrates that artificial reproductive techniques can be successful in achieving conception and ongoing pregnancy, including birth of a healthy infant, in patients where intercourse is impossible due to specific genital defects.

The First Attempt to Artificially Reproduce the Endangered Cyprinid Lake Minnow Eupallasella Perenurus (Pallas)

The standard artificial reproduction technique with the use of Ovopel (GnRH analogue) was evaluated for the critically endangered cyprinid species in Poland, the lake minnow Eupallasella perenurus. Wild spawners were angled at the beginning of the spawning season. Only females with a condition coefficient value higher than the mean were included in the experiment and then stimulated with Ovopel. Group A (n = 20) was given standard treatment (0.2 pellet kg−1 and 1.0 pellet kg−1 after 12 h), whereas group B (n = 8) was treated with a single dose of 2.0 pellets kg−1. After fertilisation with the dry method, the glutinous eggs attached to the bottom of flow-through aquaria were incubated at 17.1–18.4°C. Throughout incubation (85D°), dead eggs were removed and counted. The same was done with dead larvae, those with deformed bodies or those with empty alimentary tracts after 2 days of external feeding. Two injections of Ovopel resulted in a significantly (P ≤ 0.05) higher ovulation rate in comparison with the single dose (70 and 25% in groups A and B, respectively). The individual hatching rates were very high (98.4–100%), as was the share of good quality larvae (91.1 and 96.5% of stripped eggs in groups A and B, respectively). These results indicate that the standard propagation method used with commercially important cyprinid species can also be used to successfully breed E. perenurus.

Endometrial volume as assessed by three-dimensional ultrasound is a predictor of pregnancy outcome after in vitro fertilization and embryo transfer

Successful implantation after artificial reproductive techniques (ART) depends on multiple factors including embryo quality and endometrial receptivity. At present, endometrial thickness is used as the most important parameter for endometrial receptivity for the routine monitoring of controlled ovarian hyperstimulation. Three-dimensional (3D) transvaginal ultrasound is a relatively new technique that has become more common in the field of reproductive technologies.

Birth weight and age at menarche in patients with polycystic ovary syndrome or diminished ovarian reserve, in a retrospective cohort.

Few studies have investigated the association between subfertility in women and factors in early life such as birth weight and age at menarche, and most have produced contradictory results. In the present study, this association was investigated among women undergoing artificial reproductive techniques (ART), including IVF for reason of polycystic ovary syndrome (PCOS) or diminished ovarian reserve. Herein, PCOS included oligomenorrhoea and at least one additional symptom such as hyperandrogenism, hirsutism or polycystic ovaries on ultrasound. In most patients this was concomitant with elevated serum LH levels. Diminished ovarian reserve was defined as receiving a donated oocyte or having a low response to ovarian hyperstimulation. Among a retrospective cohort of 26 428 women diagnosed with subfertility between 1980 and 1995, three study groups and one reference group were defined using data from medical records. Women were included in the first group if diagnosed as having PCOS (n = 265). In order to define diminished ovarian reserve capacity, two groups were selected: (i) women receiving a donated oocyte (n = 98); and (ii) women having a low response (three follicles or less) to ovarian hyperstimulation in both their first and second IVF cycles (n = 351). Women with tubal obstruction formed the reference group (n = 957). In a logistic regression model, the effect of birth weight and age at menarche was examined. Information on both variables was obtained from mailed questionnaires. Birth weight did not differ significantly between the study groups and the reference group. However, PCOS patients were significantly older at menarche [OR 3.31 (2.18-5.04)]. Women receiving a donated oocyte and low responders were significantly younger at menarche [OR 2.67 (1.35-5.29) and OR 2.01 (1.26-3.20) respectively]. The fetal origins hypothesis, the association between intrauterine growth retardation and disease in adult life, could not be confirmed, though a relationship between timing of menarche and PCOS and a diminished ovarian reserve was identified. Further investigation of the effect of birth weight on fertility outcome in a prospective setting is strongly advised.

Accuracy of Ultrasound Dating Formulae in the Late Second-Trimester in Pregnancies Conceived with in-vitro Fertilization

To investigate the accuracy of ultrasound dating formulae in the late second trimester of pregnancy. A dataset of 136 singleton pregnancies conceived by artificial reproductive techniques was studied to assess the accuracy of ultrasound dating formulae in the late second trimester, and compared with early second trimester. A total of 21 published ultrasound-dating formulae were tested. For most formulae, late second trimester scans yielded results that were marginally less accurate than the early second trimester. The best performance was obtained with dating formulae based on femur length, either alone or combined with the biparietal diameter. These formulae had mean absolute errors of 3-3.5 days. Combining two or more parameters did not result in any substantial gain in accuracy. Pregnancy dating by ultrasound in the late second trimester is sufficiently accurate for routine clinical use. Formulae based on femur length appear to be at least as accurate as head measurements.

Accuracy of ultrasound dating formulae in the late second-trimester in pregnancies conceived with in-vitro fertilization.

To investigate the accuracy of ultrasound dating formulae in the late second trimester of pregnancy. A dataset of 136 singleton pregnancies conceived by artificial reproductive techniques was studied to assess the accuracy of ultrasound dating formulae in the late second trimester, and compared with early second trimester. A total of 21 published ultrasound-dating formulae were tested. For most formulae, late second trimester scans yielded results that were marginally less accurate than the early second trimester. The best performance was obtained with dating formulae based on femur length, either alone or combined with the biparietal diameter. These formulae had mean absolute errors of 3-3.5 days. Combining two or more parameters did not result in any substantial gain in accuracy. Pregnancy dating by ultrasound in the late second trimester is sufficiently accurate for routine clinical use. Formulae based on femur length appear to be at least as accurate as head measurements.

Cumulus cell apoptosis as a predictor for oocyte quality in artificial reproduction technique

To be able to predict the success of ART reliable tests for determining the quality of the oocytes are necessary. Apart from a vague morphologic assessment via microscopy a direct analysis of the oocyte quality is not possible. Because of the very close relation between the oocyte and the cumulus cells the analysis of the cumulus cells might give sufficient information on the oocyte quality. In this study we correlate the apoptotic activity of cumulus cells to the outcome of fertilized oocytes after Intracytoplasmic Sperm Injection (ICSI). 246 cumulus-oocyte-complexes from patients undergoing infertility treatment with the ICSI procedure were individually collected. The comet assay was used to determine the proportion of apoptotic cells within the cumulus population of each oocyte and correlated with oocyte fertilization and oocyte quality as well as with pregnancy outcome in 86 patients. We were able to show that high quality embryos correlate to a low rate of apoptotic cells in their corresponding cumuli. Differences regarding the pregnancy outcome were statistically not significant. Our results on cumulus cell apoptosis and embryo quality confirm other publications. To arrive at statistically proven criteria for the further development of single oocytes an increase in the number of analyzed patients is necessary.

Impact of three-dimensionally measured endometrial volume on the pregnancy rate after intrauterine insemination.

The success of artificial reproductive techniques not only depends on the quality of oocytes and spermatozoa but also on the receptivity of the endometrium. The aim of this study was to assess the role of endometrial volume measurement by three-dimensional ultrasound in predicting the pregnancy rate (PR) in women receiving controlled ovarian hyperstimulation followed by intrauterine insemination. 104 patients having intrauterine insemination (IUI) were included in this prospective trial. Ovarian hyperstimulation was performed with gonadotropins in 73 % of patients and with clomiphene citrate (CC) in 27 % of patients. Endometrial thickness, pattern and three-dimensional volume were measured immediately before insemination. In 104 IUI cycles a total of 14 clinical pregnancies were recorded (PR=13.5 %). The endometrial volume was 3.5 ml and was not significantly different in pregnant (4.0 +/- 1.5 ml) from non-pregnant women (3.4 +/- 1.9 ml). In the subgroup of women with an endometrial volume > or = 2 ml and trilaminar endometrium the pregnancy rate was 22 %, significantly higher than that in women without these two criteria (PR 6 %, p < 0.05). The negative predictive value of an endometrial volume < 2 ml for a clinical pregnancy after IUI was 96 %. Endometrial volume and thickness were significantly higher after ovarian hyperstimulation with gonadotropins (3.7 ml and 11.0 mm) than with CC (2.8 ml and 9.5 mm; p < 0.05). An endometrial volume < 2 ml at the day of insemination is associated with a poor likelihood of pregnancy. Endometrial volume measured by 3D ultrasound is a new objective parameter to predict endometrial receptivity.

A neural computational model of ICSI outcomes from sperm source and other features

Objective: With the success and increasing popularity of ICSI, accurately predicting outcomes from this artificial reproductive technique becomes highly desirable. We examined a large dataset of ICSI outcomes and associated clinical features such as sperm source, which technician performed the procedure, number of ova retrieved and maternal age, and modeled outcomes using neural computational techniques. Neural computation employs computer programs that implement mathematical models based loosely on the function of networks of biological neurons. Statistical feature extraction, allowing examination of the significance of individual features such as sperm source, was employed using Wilk’s Generalized Likelihood Ratio Test (GLRT). Design: Neural computational modeling of retrospective data. Materials/Methods: We modeled fertilization outcomes from ICSI from technician (7 different technicians performed the procedure,) sperm source (testis extraction, MESA, EEJ,) number of ova retrieved and maternal age using neUROn2++, a set of C++ programs we developed to implement neurocomputational algorithms, discriminant function and regression techniques using the Cygwin (Red Hat) GNU C++ port for Windows (Microsoft) and Visual C++ (Microsoft) distributed across Pentium (Intel) platforms. Wilk’s GLRT was implemented in neUROn2++ for statistical feature extraction. A dataset of 1070 unique ICSI outcomes was randomized into a training set of 1349 exemplars and 449 test exemplars. N1/N2 cross-validation was employed. Results: Overall model accuracy was ROC AUC 0.922 (sensitivity 86.9%, specificity 79.0%) in the training set, and 0.897 (sensitivity 82.8%, specificity 75.1%) in the test set. Feature extraction using Wilk’s GLRT in reverse regression is shown in the table. All features were significant to the model (all p <1e-5), however, with p <1e-106, technician was highly significant.TableRegression analysis of neural computational model to predict ICSI outcomes.Featurep-valueTechnician1.04e-107No. ova retrieved9.84e-45Testis extraction3.81e-21Maternal age7.70e-20MESA2.33e-10EEJ1.52e-06p-values indicate significance of the individual feature to the likelihood of fertilization with ICSI. Open table in a new tab p-values indicate significance of the individual feature to the likelihood of fertilization with ICSI. Conclusions: We modeled ICSI outcome with high accuracy (ROC AUC 0.897 in the test set) using a neural computational approach. In order to determine the significance of individual features to the model’s outcome, regression based on Wilk’s GLRT was performed. All features examined (sperm source, number of ova retrieved, maternal age and which technician performed the procedure) were statistically significant to the model’s outcome. Supported by: NIH P01 HD36289 to Dolores J Lamb, Larry I Lipshultz and Craig Niederberger.

Prevalence and outcome of pregnancies in Norwegian and Swedish women with cystic fibrosis

As patients with cystic fibrosis now live longer, more women with cystic fibrosis wish to become mothers. We looked at the prevalence and outcome of pregnancies in women with cystic fibrosis who were alive in Norway and Sweden in the year 2000. Women with cystic fibrosis aged 18 years and older attending cystic fibrosis centers in Norway and Sweden were included in the study. Information was obtained from medical records, interviews and questionnaires. One hundred and thirty-two women with cystic fibrosis were included. Sixty-one (46%) had considered or were now considering pregnancy. Forty-six (75%) of these 61 women had already conceived. Eighty pregnancies were reported, including 12 (15%) conceived by assisted reproductive techniques. There were eight (10%) induced abortions, 11 (14%) miscarriages, and one extrauterine pregnancy. The remaining 60 (75%) pregnancies resulted in live born infants. Today women with cystic fibrosis have a better chance of getting pregnant than previously reported. In this study, 75% of those who wanted to get pregnant had succeeded. Fifteen percent of pregnancies were conceived by artificial reproductive techniques. The possibility of motherhood must be considered in these patients. It is important to plan pregnancies with optimal follow up by a cystic fibrosis team.

Testicular Pathology Discovered in Candidates for Testicular Extraction of Sperm

Objectives: The development of Intracytoplasmic Sperm Injection (ICSI) made possible direct retrieval of sperm from testes for use in Artificial Reproductive Techniques. At times, the urologist is not involved in the evaluation and/or retrieval of sperm in the infertile male. We found significant testicular pathology in patients who were candidates for testicular sperm extraction. These would have been discovered with an appropriate urological evaluation. Failure to detect these entities prior to TESE would have compromised patient care and possibly lead to medical legal action.

THROMBOEMBOLIC COMPLICATIONS ASSOCIATED WITH REPRODUCTIVE ENDOCRINOLOGIC PROCEDURES

Thromboembolism as a complication of hormonal ovarian stimulation in the context of artificial reproductive techniques is rare and seems to occur when OHS is present. Although accompanied by high serum estrogen concentrations, hCG seems to play a central role in the development of OHS, which has been observed in women with 17,18-desmolase deficiency who have low estrogen levels after induction of ovulation with hGC. Although there is some evidence that hormonal ovarian stimulation with HMG, leading to elevated estrogen levels, and ovulation induction with hCG in preparation for in vitro fertilization are associated with a state of hypercoagulability, the exact role of estrogens, hCG and the physicochemical changes (fluid shift into third spaces) involved in OHS remain to be elucidated.

Limitations of reproductive medicine from an andrological/urological point of view

Since the first child was born after in-vitro fertilization in 1978, different techniques of ovarian stimulation, oocyte retrieval and embryo culture have improved significantly. The development of microinjection techniques was a mile stone in reproductive medicine. Even in cases of severe impairment of semen quality, using the Intracytoplasmic Sperm Injection (ICSI), pregnancy rates of approximately 20 % per treatment cycle can be achieved. However for using artificial reproductive techniques (ART) the female age is a major limiting factor as well as the development of premature ovarian failure (POF). Crucial for the male is the question whether or not sperm cells can be retrieved. Today an interdisciplinary approach is recommended to take care of the afflicted couple in order to address the complex problem of infertility. There is evidence that children born after microinjection techniques bare a higher risk for chromosomal aberrations compared to the normal population. However rates of major malformations seem to be identical in both groups. An evaluation of the potential risks and efforts and an extensive counseling of the infertile couple has to be taken before initiating invasive ART.

Loss of livestock breeding efficiency due to uncompensable sperm nuclear defects.

An important goal of modern analyses of semen is to elucidate the molecular traits of mammalian sperm chromatin structural abnormalities, defined here as 'uncompensable', that lead to abnormalities in fertility, pronuclear formation, early embryo quality and pregnancy outcome. Sperm with uncompensable nuclear abnormalities are able to fertilize oocytes both in vivo and in vitro; however, due to the uncompensable trait(s), the embryo development may be abnormal. Uncompensable nuclear traits can be experimentally induced in bull sperm by a mild thermal insult to the testis. Sperm nuclear morphology abnormalities seen in ejaculates 11-days post stress are likely related to molecular changes in chromatin observed 3-days post stress by the flow cytometric sperm chromatin structure assay (SCSA). The SCSA measures the susceptibility of sperm nuclear DNA to denaturation in situ. This susceptibility has been correlated with the presence of DNA strand breaks that may be derived in part by oxidative stress and possibly by a unique, abortive apoptotic mechanism. The extent of DNA denaturation is not significantly related to the level of disulfide bonding between the chromatin protamines. The use of human sperm with uncompensable nuclear traits for artificial reproductive techniques is also discussed. The goal of this research is to remove from semen doses those sperm with uncompensable nuclear traits and thereby increase male fertility potential.

Successful embryo transfer in the silver fox (Vulpes vulpes).

Surgical embryo transfer in the silver fox was investigated as part of a larger project concerning the conservation of endangered canine species using modern artificial reproduction techniques with the farmed fox as a model. The animals were chosen on the basis of synchrony in natural oestrus. The timing of ovulation and artificial insemination was determined by measuring electrical resistance in the vagina. Twenty-nine embryos were flushed from eight humanely killed donor females and transferred surgically into the uteri of eight recipients. One recipient female gave birth to two male pups 47 days after the transfer of four expanded blastocysts and one embryo at the 16-cell stage derived from a donor female flushed 10 days after artificial insemination.

Lower risks of adverse outcome in twins conceived by artificial reproductive techniques compared with spontaneously conceived twins

The outcomes of twins conceived by 136 women after medical assistance (MA) such as ovulation induction with or without assisted reproductive techniques, and twins conceived spontaneously (SP) by 72 women were compared. All 208 women were monitored from < 20 weeks gestation; they all delivered at > or = 24 weeks gestation. The chorionicity of the placenta was diagnosed antenatally and confirmed after delivery. There were 10 perinatal deaths; the physical and neurological status of the remaining 406 infants was assessed at 1 year of corrected age. There were no differences in gestational age at birth, the birth weights of the larger and smaller twins, the birth weight discordance, or the incidence of life-threatening major malformations between groups. Adverse infant outcomes, such as death, cerebral palsy and mental retardation occurred in nine (3.3%) of 272 MA twins compared with 12 (8.3%) of 144 SP twins (P < 0.05). The placenta was monochorionic in only three (2.2%) of 136 MA twin pregnancies compared with 41 (57%) of 72 SP twin pregnancies (P < 0.001). Of the 21 infants with adverse outcomes, nine had monochorionic placentas. Thus, the risk of an adverse outcome was 2.8-fold higher (95% confidence interval (CI) 1.2-6.4) in monochorionic twins than in dichorionic twins (10 versus 3.7%; P < 0.05). There was no difference in the incidence of adverse infant outcomes between SP (4.8%) and MA (3.4%) twins with dichorionic placentas. These findings suggest that ovulation induction in itself was not associated with an adverse outcome of twin pregnancies. The lower frequency of monochorionic placentas in MA twins may have been responsible for the lower risk of an adverse outcome in MA twins.

Induction of spermatogenesis in isolated hypogonadotrophic hypogonadism with gonadotrophins and early intervention with intracytoplasmic sperm injection

Idiopathic hypogonadotrophic hypogonadism (IHH) is a potentially correctable cause of male infertility. However hormonal treatment is usually a slow process and artificial reproductive techniques such as intracytoplasmic sperm injection (ICSI) might be resorted to before full testicular response has been achieved. We report here an unusual variant of IHH of post-pubertal onset in which early intervention with ICSI was attempted. Our patient was 37 years old and presented with male infertility due to azoospermia and undetectable serum gonadotrophin concentrations. He had an apparently normal pubertal development, a testicular volume of 8 ml, normal pituitary-thyroid and pituitary-adrenal function, as well as normal computerized tomographic appearance of the sella region. A combination of human chorionic gonadotrophin (HCG) and menopausal gonadotrophins (HMG) was administered. Spermatozoa were first detected in the semen after 3 months and reached a concentration of approximately 2x10(6)/ml after 9 months. ICSI was attempted at this point; the spermatozoa had good fertilizing ability and three embryos were obtained and replaced. Unfortunately no pregnancy resulted. Treatment with 5000 IU HCG and 150 IU HMG three times per week was continued and sperm counts rose rapidly thereafter to reach 28.3x10(6)/ml after 16 months of injections. His wife conceived naturally during this period and the pregnancy is now in the second trimester. This case illustrates the good prognosis of the rare patient with IHH of post-pubertal onset when treated with gonadotrophins, and suggests that ICSI procedures should be delayed until final testicular maturation is attained.