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PubMed1990

Suboptimal laboratory conditions can affect pregnancy outcome after embryo transfer on day 1 or 2 after insemination in vitro.

We have demonstrated that the replacement of pronuclear stage human embryos on the day after insemination in IVF-ET results in a pregnancy rate as good as, if not better than, the rate after ET on day 2. The pregnancy rate appears to be influenced by the culture environment used for the gametes. We would recommend that ET be undertaken as soon as possible after insemination on day 1 so that if suboptimal culture conditions are present, they do not have sufficient time to affect the viability of the embryos and reduce the subsequent chance of pregnancy after replacement. If culture conditions have been optimized there is no difference in pregnancy rate after embryo transfer on day 1 or day 2 and one can then replace embryos on either day and expect the pregnancy rate obtained in either case to be similar.

Embryo Transfer↗
PubMed1992

Human embryo biopsy on the 2nd day after insemination for preimplantation diagnosis: removal of a quarter of embryo retards cleavage.

OBJECTIVE: To assess any reduction in viability and development in vitro after biopsy of a quarter of the cells of human embryos on day 2 after insemination. DESIGN: A prospective study in which normally fertilized surplus embryos of good morphology with two to eight cells approximately 48 hours after insemination were randomly allocated to a control or biopsied group, respectively. SETTING: In vitro fertilization (IVF) unit and laboratories of the Hammersmith Hospital, Institute of Obstetrics and Gynaecology, London University. PATIENTS, PARTICIPANTS: One hundred twenty-nine embryos from 28 infertile IVF patients. INTERVENTIONS: Follicular aspiration by ultrasound-guided transvaginal puncture and embryo biopsy by micromanipulative procedures. MAIN OUTCOME MEASURE(S): Pyruvate uptake and cell number at the blastocyst stage. RESULTS: Embryo biopsy did not have an adverse effect on either the proportion developing to the blastocyst stage (50% [32 of 64] and 47.7% [31 of 65] for the control and biopsied groups, respectively) or embryo viability, measured indirectly through pyruvate uptake. However, the proportion of embryos that reached the morula stage after day 4 (retarded embryos) was significantly higher (44%, 11 of 25 versus 8.7%, 2 of 23) in the biopsied group. The total number of cells (29.6 +/- 3.1 versus 62.4 +/- 4.7), numbers of inner cell mass (7.7 +/- 2.2 versus 24.5 +/- 1.4) and trophectoderm (24.0 +/- 5.2 versus 45.0 +/- 6.4) cells, and the inner cell mass:trophectoderm ratio (34.7 +/- 7.9 versus 59.5 +/- 11.7) were strikingly reduced at the blastocyst stage in the biopsied group. This reduction was greater in embryos that reached the morula stage after day 4. CONCLUSIONS: More investigation is needed to assess whether the detrimental effects observed were because of the biopsy method used in this study or to a high sensitivity of human embryos at early stages to manipulation in vitro.

Biopsy↗
PubMed1995

High levels of sperm-associated antibodies impair human spermoolemma interaction after subzonal insemination.

OBJECTIVE: To investigate the impact of sperm-associated antibodies in the fertilization process beyond the zona pellucida in human oocytes. DESIGN: Subzonal insemination (SUZI) was performed with antibody-coated spermatozoa from patients with autoimmune infertility. Sperm parameters and antibody binding were analyzed after Percoll selection and were compared with SUZI outcome. PATIENTS: Patients (n = 29) with an immunobead test higher than 60% for immunoglobulin (Ig)G, IgA, or both classes were included in the study (38 attempts). Twenty-six patients had previous IVF failures, and the semen characteristics for three were unsuitable for IVF. RESULTS: Diploid fertilization occurred in 52.6% of cycles and 16.0% of oocytes (n = 263). The fertilization rate was inversely correlated to the proportion of spermatozoa coated with IgG in the Percoll-selected sperm suspension. Three of the 16 attempts in which > 90% of the spermatozoa were coated with IgG resulted in a low diploid rate of 4.1%. Fertilization was obtained in 18 of the 22 attempts in which < 90% of spermatozoa were IgG coated, with a diploid fertilization rate of 22.8%. Association of both classes of antibodies (IgG and IgA) further impaired the fertilization rate. Twenty ETs gave four pregnancies: one biochemical, one ongoing, and two giving birth to healty babies (1 singleton and 2 twins). CONCLUSIONS: Subzonal insemination is a potential solution for achieving fertilization in cases of IVF failure because of sperm autoimmunity. However, IgG inhibits in a dose-dependent manner the fusion of gametes' membranes. Though the level of IgA antibodies does not appear as critical as that of IgG antibodies, association of both classes of Igs impairs the probability of fertilization.

Adult↗
PubMed2000

Artificial insemination using cryopreserved sperm in the silkworm, Bombyx mori.

We report in this paper that female moths artificially inseminated with cryopreserved sperm (-196 degrees C) could oviposit eggs when the sperm was preserved for 356days, and that the fertilization rate and the number of eggs laid were almost equivalent to those obtained in normally mated moths. The optimal cooling rate for sperm freezing was 5-65 degrees C/min for maintaining a high fertility of sperm. The simple and reliable method of cryopreservation was to put the semen first in a deep freezer at -80 degrees C and thereafter put them in liquid nitrogen. When female moths of 'white 2' egg-color mutant strain were inseminated with a mixture of frozen-thawed sperm from males of normal-colored egg strain and non-frozen sperm from males of the 'white 2', female moths deposited a majority of 'white 2' eggs and a very small number of eggs of normal color. The result shows that there was a competitive fertilization of sperm between the two strains of the silkworm, and that sperm fertility was reduced to a considerable extent by freezing at -196 degrees C. These results may contribute not only to basic studies on fertilization in Lepidoptera but also to the development of long-term preservation procedure of genetic resources by using cryopreserved sperm of Bombyx mori.

Journal Article↗
PubMed1988

Effects of single deep insemination on transferable embryo recovery rates in superovulated dairy cows.

Thirty superovulated Friesian lactating cows were randomly assigned to two groups. Group I donors were inseminated with one unit of semen deposited into the uterine body at 8, 20 and 32 h after the onset of estrus. Group II donors were inseminated with one unit of semen deposited deep into the uterine horns at 15 h after the onset of estrus. Neither the mean rates of fertilized ova nor the mean rates of transferable embryos were different between treatments (P > 0.05).

Journal Article↗
PubMed1997

Fertility and ovum fertilization rate after laparoscopic or transcervical intrauterine artificial insemination of oxytocin-treated ewes.

Based on our previous work, we found that exogenous oxytocin induces uterine tetany and cervical dilation, and permits transcervical access to the uterus. However, the oxytocin does not reduce sustained sperm transport from the uterus to the oviducts. Thus, we hypothesized that exogenous oxytocin may be a useful adjunct to transcervical intrauterine AI procedures for sheep: two experiments were conducted to test our hypothesis. In Experiment 1, purebred ewes (n = 75/group) were artificially inseminated intrauterine with either laparoscopic or oxytocin-transcervical (i.e., 200 USP units of oxytocin 30 min before AI) procedures. At 54 h after progestogenated pessaries were removed, ewes were inseminated with 200 x 10(6) sperm/0.25 ml of fresh, extended semen, which was collected from a purebred ram of the corresponding breed. Pregnancy rate was greater (P < 0.05) after laparoscopic (37.5%) than after transcervical AI (0%). Because of the disappointing results of Experiment 1, Experiment 2 was conducted to determine whether oxytocin or the AI procedure per se reduced ovum fertilization rate. Treatments were designed in a 2 x 2 factorial arrangement. At 60 h after norgestomet implant removal and 10 min before either laparoscopic or transcervical (cervical in a saline group) AI with 100 x 10(6) sperm/0.25 ml, ewes (n = 10/group) received an intravenous injection of either isotonic saline or 200 USP units of oxytocin. Fertilization rate, which was determined 72 h after AI, was greater (P < 0.05) after laparoscopic than after transcervical/cervical AI (92.5 vs 28%), but oxytocin treatment did not affect fertilization rate. The results indicate that exogenous oxytocin did not reduce ovum fertilization rate, but the transcervical AI procedure per se seemed to reduce fertilization rate.

Journal Article↗
PubMed2001

Effect of time of artificial insemination on fertility of progestagen and PMSG treated indigenous Greek ewes, during non-breeding season.

A total of 2567 indigenous Greek ewes (Chios, Vlachiki and VlachikixChios breeds) were used to determine the optimum time for insemination, following synchronization of oestrus with MAP-impregnated intravaginal sponges and PMSG during non-breeding season. Within each breed group, the ewes were divided into three subgroups and submitted to a double blind cervical artificial insemination 48 and 60h (subgroup I), 60 and 72h (subgroup II) and 48 and 72h (subgroup III) after sponge withdrawal. From the results of the present investigation it can be concluded that the conception rate in the Chios island breed is better than that in the VlachikixChios breed, with the latter being better than that in the Vlachiki breed. Using fixed time for AI, a better conception rate is obtained when applied 48 and 72h after sponge withdrawal for Chios and ChiosxVlachiki breeds, while for Vlachiki breed a better conception rate is obtained when fixed AI is applied 48 and 60h after sponge withdrawal.

Journal Article↗
PubMed1991

The impact of sonographic assessment of the endometrium and meticulous hormonal monitoring during natural cycles in patients with failed donor artificial insemination.

The present study was undertaken to determine whether the sonographic characteristics of the endometrium in the proliferative phase of the natural cycle combined with meticulous hormonal monitoring would have an impact on the prediction of conception. Fourteen women with regular ovulatory cycles were examined daily, from day 8 of the cycle, by transvaginal ultrasound through 16 unstimulated cycles. All patients had had at least four previous unsuccessful donor artificial insemination cycles scheduled by basal body temperature records. During the study, donor artificial insemination was meticulously timed by hormonal and sonographic monitoring. Four clinical pregnancies occurred with a success rate of 29% per patient, or 25% per cycle. The endometrial thickness at the mid-proliferative phase (day 8) was significantly smaller in patients who conceived in comparison with patients who failed to conceive (0.33 +/- 0.07 cm vs. 0.59 +/- 0.03 cm; p < 0.01). The endometrial thickness on the day of the luteinizing hormone (LH) peak was not significantly different between the two groups. However, the amount of endometrial growth between day 8 of the cycle and the day of the LH peak was significantly greater in conception than non-conception cycles (0.55 +/- 0.05 cm and 0.24 +/- 0.06 cm; p < 0.01). No difference in hormonal parameters was observed either on day 8 of the cycle or on the day of the LH peak between conception and non-conception cycles. On the day of the LH peak, an endometrium with typical triple line appearance and thickness of 0.6 cm or more was seen significantly more often in conception than non-conception cycles (p < 0.05). No pregnancy occurred with an endometrium < 0.6 cm at the time of ovulation. The presence of the favorable endometrial pattern in the periovulatory phase was associated with a positive predictive value for pregnancy of 50% (sensitivity of 100% and specificity of 67%), whereas the negative predictive value of the hyperechoic pattern at the time of ovulation was 100%. Our data show negative correlation between early growth of the endometrium and pregnancy in unstimulated cycles, suggesting that the amount of endometrial growth during the cycle up to ovulation may have an important role in implantation. The sonographic image of the endometrium in natural cycles provides useful information about the possible outcome of the treated cycle.

Journal Article↗
PubMed1999

Insemination at the second of two induced oestrous periods in anoestrous dairy cows increases conception rates to first service.

AIM: To determine whether mating previously anoestrous cows on their second, rather than first, oestrus post pm-turn could increase conception rates to that service. METHODS: Cows diagnosed as anovulatory anoestrus were allocated to two groups, starting treatment on Day -17 (OES2) or on Day -7 (OESI) where the date of the planned start of mating = Day 0. All cows were treated with intravaginal progesterone (CIDR device) for 6 days and an injection of 1 mg oestradiol benzoate 24 h after removal of the device. Cows in the OES2 group were also injected with prostaglandin F2alpha on Day 0. RESULTS: During the first 7 days of mating, 61.8% (207/335) and 88.2% (328/372) of cows in the OES2 and OESl groups, respectively, were inseminated on detection of oestrus (p <0.001). Oestrus without ovulation occurred in 1.5% of cows in the OES2 group and 6.2% of cows in the OESl group during this period (p <0.01). Of those cows inseminated in the first week of mating, conception rates were 54.0% in the OES2 group and 45.5% in the OESl group (p = 0.05). There was no significant difference in median interval from start of mating to conception between the two groups (24 and 22 days for OES2 and OESl, respectively, p >0.05). CONCLUSION: Mating previously anoestrous cows on their second, rather than first oestrus post partum increased conception rates to that service. However, median intervals from start of mating to conception were not altered.

Journal Article↗
PubMed2000

The value of gonadotrophin releasing hormone analogue in an intrauterine insemination (IUI) programme.

Gonadotrophin releasing hormone analogue (GnRHa) is widely used in in-vitro fertilisation programmes and has no doubt led to a significant improvement in the pregnancy rate as well as cycle planning. However, its use in intrauterine insemination (IUI) programmes remains controversial. This is a retrospective review of patients who received IUI treatment at our assisted conception unit, during a 12-month period from August 1997 to July 1998. Fifty-two patients underwent a total of 85 cycles. Six cycles were abandoned, five due to excessive response and one for continuous bleeding. They all received Nafarelin nasal spray from day 2 until ovulatory HCG was administered. Metrodin injection was given from day 3. There were 18 clinical pregnancies. Seventy-nine cycles were completed giving a pregnancy rate of 22.8% per completed cycle. There were five (27.8%) multiple pregnancies. The use of GnRHa in our programme enabled us to achieve a good cycle control as well as flexibility in the timing of insemination such that weekend work was avoided. We conclude that the use of GnRHa facilitates the efficiency of an IUI programme.

Journal Article↗
PubMed1997

Length of the follicular phase, time of insemination, coital rate and the sex of offspring.

The penetrability of cervical mucus improves over the follicular phase. When the length of the follicular phase varies due to variation in the timing of the luteinizing hormone surge, mucus penetrability will also improve as the phase lengthens. As selection for Y spermatozoa decreases with improvements in mucus penetrability, sex ratios at conception should decline in longer follicular phases. Sex ratios should also decline as the time of insemination approaches ovulation unless hormonally-induced improvements in penetrability are reduced by the debris left by earlier inseminations.

Cervix Mucus↗
PubMed2002

Spectral karyotyping of fresh, non-inseminated oocytes.

The object of this study was to determine the mechanisms producing aneuploidy in female meiosis I by analysing the whole chromosome complement of human non-inseminated and unfertilized fresh oocytes. For this purpose, 131 fresh oocytes were obtained from 16 oocyte donors (24-48 years old). These oocytes were fixed immediately after retrieval and 47 good quality metaphases from 13 donors were analysed by spectral karyotyping to identify all 23 chromosome types. The data was divided into two maternal age groups, 24-34 (n = 31) and > or = 35 years (n = 16). More non-disjunction (13 and 25%), unbalanced predivision (10 and 44%, P < 0.01) and balanced predivision (6 and 62%, P < 0.001) events were found in the older group of oocytes. There was an increase in balanced predivision with decreasing chromosome size (P < 0.001). The present results are the first obtained in fresh oocytes where all chromosomes were specifically identified, and support previous theories that predivision of chromatids is a major factor causing aneuploidy. Previous reports with inseminated, non-fertilized oocytes fixed > or = 24 h after retrieval suffered from artefactual predivision of chromatids triggered by in-vitro ageing of oocytes.

Adult↗
PubMed1996

Fertilization, embryonic development and implantation of mouse oocytes with one or two laser-drilled holes in the zona, and inseminated at different sperm concentrations.

The zonae pellucidae of mouse oocytes were photo-ablated by an ultraviolet laser (337.1 nm) to create one or two 10 microm holes. The pulse energy used was approximately 3 microJ/s, with a frequency of 10 pulse/s. These laser zona-drilled (LZD) oocytes with one hole (LZD1) or two holes (LZD2) and the zona-intact controls were inseminated with spermatozoa at standard concentrations of 5x10(4), 5x10(5), 1x10(6) and 2x10(6)/ml. Fertilization was significantly improved at all sperm concentrations in LZD1 and LZD2 oocytes as compared to the controls. However, there was no significant difference in fertilization rates between LZD1 and LZD2. LZD2 produced significantly higher numbers of blastocysts at day 5. Hatching was significantly enhanced in the presence of either one or two holes in the zona. Polyploidy was generally absent, except in LZD2 oocytes (1%) inseminated at higher sperm concentrations. Differential cell counts of expanded LZD blastocysts were similar to those of the controls. Significantly fewer LZD2 blastocysts implanted and produced viable fetuses than LZD1 and control blastocysts. Morphological abnormalities of the fetuses were absent in all three groups. Laser zona-drilling using the ultraviolet laser was shown to be fast, efficient and safe.

Animals↗
PubMed1989

The frequency of chromosome anomalies in human unfertilized oocytes and uncleaved zygotes after insemination in vitro.

Data on first trimester human abortions have shown chromosome aberrations in approximately 50% of the cases, most of which were trisomies. More than 80% of these trisomies have been attributed to an error in the oocyte. In this study we investigated the frequency of chromosome anomalies in 44 human unfertilized oocytes and 13 uncleaved zygotes after insemination in vitro. The oocytes were aspirated from preovulatory follicles in parous volunteer donors and patients participating in an embryo replacement programme. Thirty cases could be analysed, 18 of which (60%) carried a chromosome abnormality. There were two hyperhaploidies (6.7%), four hypohaploidies (13.3%), three polyploidies (10%), six with structural aberrations (20%) and five diploidies (16.7%). The incidence of these abnormalities, except for the structural anomalies, was similar to that reported previously. The higher frequency of structural aberrations in the present study may be related to delayed cytogenetic analysis for up to 48 h after insemination.

Abortion, Spontaneous↗
PubMed1991

Presentation of six pregnancies established by sub-zonal insemination (SUZI).

Clinical trials were undertaken to evaluate sub-zonal insemination (SUZI) for the procurement of fertilization in infertile couples. We present here the six pregnancies resulting from this work. Insemination of multiple sperm was used, with a mean of 3.7 +/- 2.1 (SD) per oocyte. One hundred and thirty patients presented either with previous failures of fertilization in vitro and/or with severe seminal defects, including oligo-, astheno-, oligo-astheno- or necro-zoospermia. Forty-one (31.5%) patients achieved fertilization and 39 had replacement of conceptuses. Six patients achieved a pregnancy (4.6% overall, 15.4% per replacement), four have delivered and two have miscarried. The implantation rate was nine conceptuses from a total of 61 replaced (14.8%); and of the six women who conceived, nine of 11 replaced conceptuses implanted. Of the oocytes recovered, 539 were used for SUZI, of which 69 (12.8%) fertilized. Three singleton pregnancies resulted, two from patients having one conceptus replaced, the other from the replacement of three conceptuses. Two twin pregnancies were produced, both in patients having two conceptuses replaced. The sixth pregnancy, a triplet, occurred in a patient having three conceptuses replaced, one conceived after standard in-vitro fertilization (IVF), the other two after SUZI. Two sets of non-identical twins, both one male and one female, a singleton female and female (non-identical) triplets have been born, normal and healthy.

Adult↗
PubMed1993

Ultrasound evidence for variability in the size and development of normal human embryos before the tenth post-insemination week after assisted reproductive technologies.

Differences in human chorionic gonadotrophin (HCG), average chorionic sac diameter, embryo/fetus crown-rump length and biparietal diameter were determined in 107 singleton pregnancies delivered after 34 weeks, whose post-insemination age was precisely known as a result of in-vitro fertilization (n = 28) or gamete intra-Fallopian transfer (n = 79). Crown-rump lengths were interpreted in relation to the developmental stage of the embryo. A 7-fold to 10-fold difference in HCG levels was observed on post-insemination days 13-16. A 2-fold to 3-fold difference occurred in average chorionic sac diameter on days 25-36. A 2-fold difference occurred in crown-rump length on seven of 10 days prior to day 44. Biparietal diameter differed by no more than 42% from day 57-68 and 20% after day 68. Chorionic sac diameter, crown-rump length, and biparietal diameter were also related to newborn weight. The results suggest firstly that marked differences occur in the rate of early human development, secondly, that the differences occur prior to day 27 when observable cardiac activity begins, and thirdly, that the differences are minimized after day 68 when the embryonic period of development is completed.

Anthropometry↗
PubMed1997

Monoclonal antibodies against boar sperm zona pellucida-binding protein AWN-1. Characterization of a continuous antigenic determinant and immunolocalization of AWN epitopes in inseminated sows.

Boar spermadhesin AWN-1 is a sperm surface-associated 14.7-kDa lectin and a major protein of porcine seminal plasma. AWN-1 binds to beta-galactosides and to porcine zona pellucida glycoproteins, suggesting that this protein might play a role in the primary binding of spermatozoa to the egg's external glycoprotein matrix. We have produced a collection of murine monoclonal antibodies against purified AWN-1. Five monoclonal antibodies recognized sequential antigenic determinants. All these epitopes were located at the C-terminal region of AWN-1 (residues 109-123) by competitive ELISA using overlapping synthetic peptides that cover the complete 133 amino acid sequence of the lectin. In a structural model of spermadhesin AWN-1, the polypeptide stretch 109-123 is fully solvent-exposed, providing a reasonable explanation for its high immunogenicity. In addition to epitope mapping, we have employed anti-AWN monoclonal antibodies for immunolocalization of the protein in the genital tract of inseminated sows. Clusters of AWN epitopes were occasionally found attached to the epithelium of the uterotubal junction and the adjacent lower isthmus. However, neither AWN-1 nor other seminal plasma proteins were found in the isthmic fluid collected 10-26 h after insemination. These results suggest that the whole amount of seminal plasma proteins are absorbed by the epithelium of the female genital tract, supporting the claim that removal of seminal plasma components from spermatozoa might be a major event in both in vitro and in vivo sperm capacitation.

Animals↗
PubMed2003

Soay rams target reproductive activity towards promiscuous females' optimal insemination period.

Female promiscuity is thought to have resulted in the evolution of male behaviours that confer advantages in the sperm competition that ensues. In mammalian species, males can gain a post-copulatory advantage in this sperm 'raffle' by inseminating females at the optimal time relative to ovulation, leading to the prediction that males should preferentially associate and copulate with females at these times. To the best of our knowledge, we provide the first high-resolution test of this prediction using feral Soay sheep, which have a mating system characterized by male competition for access to highly promiscuous females. We find that competitive males time their mate guarding (and hence copulations) to occur close to the optimal insemination period (OIP), when females are also increasingly likely to 'cooperate' with copulation attempts. Subordinate males practice an alternative mating tactic, where they break the integrity of the consort pair and force copulations on females. The timing of these forced copulations is also targeted towards the OIP. We thus provide quantitative evidence that female promiscuity has resulted in the evolution of reproductive strategies in which males 'load' the sperm raffle by targeting their mating activity towards female OIPs, when the probability of sperm-competition success is at its greatest.

Animals↗