Effect of transferring a morphologically impaired embryo with a good quality embryo on the pregnancy and implantation rates.

OBJECTIVE: This study aimed to investigate the effect of transferring embryos with different qualities on pregnancy and implantation rates. PATIENTS AND METHODS: In a retrospective multi-center study we analyzed 761 patients aged ≤ 35 years who had an elective transfer of one or two embryos. Embryos were scored morphologically by their developmental stage into good "A" and impaired "B" embryos. Pregnancy and implantation rates were compared between patients who had a transfer of: one grade "A" embryo; two grade "A" embryos, two embryos one grade "A" plus one grade "B" embryos; one grade "B" embryo and two grade "B" embryos. RESULTS: Higher pregnancy and implantation rates were observed in patients who had received one embryo of grade "A" (34.6%) and two grade "A" embryos (45.2%, 25.85% respectively), compared to patients who received two embryos, one of grade "A" plus one of grade "B" (25%, 13.77% respectively). CONCLUSIONS: Transferring a morphologically and developmentally impaired embryo, significantly lower the implantation chance of the good quality embryo.

Quality of human spermatozoa: relationship between high-magnification sperm morphology and DNA integrity.

The aim of this work is to establish the relationship between the morphology of Intracytoplasmic Morphologically Selected Sperm Injection (IMSI)-selected spermatozoa and their DNA integrity. The 45 ejaculates were randomly distributed into three treatment groups: normozoospermic, oligoasthenozoospermic and oligoasthenotheratozoospermic samples. The evaluation of DNA integrity was performed using the sperm chromatin dispersion test. It was established that DNA integrity of spermatozoa is strongly dependent on ejaculate quality (P < 0.05). The count of spermatozoa with nonfragmented DNA in normozoospermic samples was high and independent from IMSI-morphological classes (Class 1 versus Class 3, respectively) (P > 0.1). With decreased ejaculate quality, the percentage of spermatozoa with nonfragmented DNA decreased significantly (P < 0.05) independent from morphological class. Nevertheless, the rate of IMSI-selected spermatozoa with fragmented DNA within of Class 1 in normozoospermic (Group 1), in oligoasthenozoospermic (Group 2) and in oligoasthenotheratozoospermic (Group 3) samples was 21.1%, 31.8% and 54.1%, respectively. In conclusion, there is a direct relationship between morphological parameters of spermatozoa and their DNA integrity. However, the IMSI technique alone is not enough for the selection of spermatozoa with intact nuclei.

Vitrification of human ICSI/IVF spermatozoa without cryoprotectants: new capillary technology.

The aim of this study was to develop and to test the standardized aseptic technology of permeable cryoprotectant-free vitrification of human spermatozoa in capillaries (for intracytoplasmic sperm injection [ICSI] or in vitro fertilization [IVF]). To test the effect of vitrification on basic sperm parameters, each of 68 swim-up-prepared ejaculates from oligo-astheno-terato-zoospermic patients were aliquoted and distributed into 3 groups: 1) nontreated control, 2) 10 µL of spermatozoa cryopreserved by slow conventional freezing with glycerol-contented medium, and 3) 10 µL of spermatozoa vitrified in 50-µL plastic capillaries in culture medium with 0.25 M sucrose. Spermatozoa motility (1, 24, and 48 hours after warming), plasma membrane integrity, acrosomal integrity, and spontaneous capacitation-like changes were determined after warming. Aseptic cryoprotectant-free vitrification showed a significantly stronger cryoprotective effect compared with conventional freezing. One hour after warming, motility, plasma membrane integrity, and acrosomal integrity were significantly higher than is observed for conventionally frozen spermatozoa (28% vs 18%, 56% vs 22%, and 55% vs 21%, respectively; P < .05), although lower than in fresh spermatozoa (35%, 96%, and 84%, respectively; P < .05). Capacitation-like changes did not differ significantly between vitrified and conventionally frozen samples (8% vs 9%, respectively; P > .1) (2% in fresh spermatozoa). The newly developed technology of aseptic vitrification of human spermatozoa in capillaries can effectively preserve these cells from cryo-injures. Spermatozoa, vitrified by this technology, are free from seminal plasma owing to swim-up preceding vitrification and are free from permeable cryoprotectants. They are ready for further use immediately after warming without any additional treatment. Therefore, the reported technology has a great potential for use in ICSI/IVF.

Cryoprotectant-free vitrification of human spermatozoa in large (up to 0.5 mL) volume: a novel technology.

BACKGROUND: The aim of this study was to develop and to test the aseptic technology of cryoprotectant-free vitrification of human spermatozoa in large volume (for intrauterine insemination). Spermatozoa, vitrified by this technology, are free of permeant cryoprotectants and are ready for further use immediately after warming without any additional treatment (centrifugation or separation in the gradient for removal of cryoprotectant). METHODS: Each of 52 swim up-prepared ejaculates were divided into three aliquots and distributed into three treatment groups: Group 1: non-treated control; Group 2: spermatozoa cryopreserved by slow conventional freezing with glycerol-containing medium, and Group 3: spermatozoa vitrified in 0.5 mL insemination "French" straws in culture medium with 0.25 M sucrose. Sperm motility 1, 24 and 48 hours after warming, plasma membrane integrity and capacitation-like changes (spontaneous "cryo-capacitation" and acrosome reaction) were assessed after freezing-thawing. RESULTS: In contrast to conventional freezing, spermatozoa vitrified with aseptic cryoprotectant-free technology displayed superior functional characteristics. The motility rate, integrity rates of cytoplasmic, and acrosomal membranes were significantly higher after vitrification than after conventional freezing (76% vs 52%, 54% vs 28% and 44% vs 30%, respectively) (p < 0.05). However, there were no differences between vitrification and conventional freezing in the presence of glycerol in terms of percentage of spermatozoa expressing CTC-capacitation pattern (11% vs 10%, respectively) (p > 0.1). CONCLUSIONS: A basic protection from cryo-injury can be achieved for human spermatozoa using the novel technology of aseptic cryoprotectant-free vitrification in large volumes.

Effect of follicle-stimulating hormone on sperm quality and pregnancy rate.

AIM: To evaluate the possible links between ultrastructural sperm quality and the clinical pregnancy rate in infertile males treated with FSH before intracytoplasmic sperm injection (ICSI). METHODS: Forty-four infertile males with idiopathic oligo-asthenozoospermia were randomly allocated to the treated (n=24) and non-treated (control, n=20) groups. Semen analysis was carried out by light and transmission electron microscopy (TEM) before and 12 weeks after FSH therapy. ICSI was performed in all couples. RESULTS: TEM revealed a significant improvement in sperm quality after FSH treatment, particularly in men with their partners achieving clinical pregnancy. The pregnancy rate was 33 % in the treated group and 20 % in the control. CONCLUSION: RESULTS highlight a positive role of FSH therapy in infertile males before ICSI, which was correlated with an increased pregnancy rate in treated couples. We believe that improved sperm ultrastructure after FSH therapy could positively influence the quality and early stage of embryo development, thereby increasing the probability of embryo implantation.

Influence of acupuncture on idiopathic male infertility in assisted reproductive technology.

The clinical effects of acupuncture on idiopathic male infertility in sperm parameter and on therapeutic results in assisted reproductive technology were investigated. 22 patients failed in intracytoplasmic sperm injection (ICSI) with idiopathic male infertility were treated with acupuncture twice weekly for 8 weeks, followed by ICSI treatment again. The sperm concentration, motility, morphology, fertilization rates and embryo quality were observed. Quick sperm motility after acupuncture (18.3% +/- 9.6%) was significantly improved as compared with that before treatment (11.0% +/- 7.5%, P < 0.01). The normal sperm ratio was increased after acupuncture (21.1% +/- 10.4% vs 16.2% +/- 8.2%, P < 0.05). The fertilization rates after acupuncture (66.2%) were obviously higher than that before treatment (40.2%, P < 0.01). There was no significant difference in sperm concentration and general sperm motility between before and after acupuncture. The embryo quality after acupuncture was improved, but the difference between them was not significant (P > 0.05). Acupuncture can improve sperm quality and fertilization rates in assisted reproductive technology.

[The implantation receptive luteal phase of the endometrium. On the current status of molecular and cell biology research]. / Die implantationsgerechte Lutealphase des Endometriums. Zum Stand der molekularen und zellbiologischen Forschung.

The biological aim of the differentiation and maturation of endometrial tissue compartments during any menstrual cycle is the achievement of suitable conditions for blastocyst implantation and the establishment of pregnancy. Infertility and early embryonic loss are frequently caused by insufficient endometrial differentiation. Even any incomplete receptivity stage of the luteal phase endometrium will prevent attachment and implantation. We have studied the physiological changes throughout an endometrial cycle to elucidate causes of endometrial insufficiency leading to subfertility or infertility. Up to now, the histological changes described by Noyes et al. are understood as classical diagnostic approaches. However, evidence is accumulating that molecular deficits of endometrial differentiation are by no means detectable histologically, and consequently ask for the research on new diagnostic methods and parameters. There are histochemical localizations of specific protein molecules, adhesion molecules and cytokines, which permit by far more detailed and significant molecular analyses than any classical morphological means could yield. Moreover, there are convincing arguments to use further biochemical assessments on proteins of the uterine secretions as specific diagnostic parameters. The electrophoretical resolution presents typical protein patterns, which in turn can be interpreted as characteristic reflexions of the functional phases of the endometrial cycle. What is demonstrated as the so-called adequate luteal phase protein pattern clearly is the product of the receptive endometrium, reflecting the "implantation window". This is established already two days after ovulation and persists usually eight further days, if the endometrial cycle is undisturbed (15th to 24th day of the cycle).

Impact of recombinant follicle-stimulating hormone and human menopausal gonadotropins on in vitro fertilization outcome.

OBJECTIVE: To investigate possible differences between using recombinant FSH (rFSH) and hMG for ovarian stimulation in IVF/intracytoplasmic sperm injection (ICSI) cycles. DESIGN: Parallel group design. Prospective, randomized clinical study. SETTING: A tertiary care infertility clinic. PATIENT(S): A total of 578 patients of our IVF/ICSI routine were recruited. INTERVENTION(S): Treatment with hMG was used for 282 patients (282 cycles), whereas 296 patients (296 cycles) were treated with rFSH. The number of cycles leading to an embryo transfer were 248 and 259, respectively. MAIN OUTCOME MEASURES: Primary: clinical pregnancy rate. Secondary: treatment days, total dose of gonadotropin administered, number of oocytes retrieved, number of mature oocytes, and embryo quality. RESULT(S): Of the cycles with embryo transfer, the pregnancy rates were 30.1% and 32.3% in the rFSH and the hMG groups, respectively. This difference is not statistically significant (P=0.798). Treatment with rFSH resulted in a significantly higher number of recovered oocytes compared with the hMG group but was also associated with a higher number of ampoules needed to reach the criterion for hCG administration. No significant differences were found with regard to the number of mature oocytes, the number of treatment days, and the embryo quality. CONCLUSION(S): In terms of the clinical pregnancy rate, no significant differences between the two stimulation regimens can be stated.

Reproductive outcome of 32 patients with primary or secondary infertility and uterine pathology.

We did a retrospective study on 102 patients who had a laparoscopy and a hysteroscopy during investigations for primary or secondary infertility. 32 of the 102 patients had uterine pathology. Seven of them had septate uteri, eight had uterine synechiae, another six had uterine fibroids, four had a bicornuated uterus, while the remaining had either a combination of all or other uterine anomalies. After surgical treatment of these conditions ten women conceived and five pregnancies including one twin pregnancy resulted in term deliveries.

[Are there any advantages in immediate assisted fertilization compared with differed fertilization?]. / La fécondation assistée immédiate (FAI) présente-t-elle des avantages par rapport à la fécondation différée (FAD)?

OBJECTIVES: To demonstrate a difference between the two assisted fertilization procedures. Can either of these procedures be preferred on the basis of preoperative parameters or criteria? A protocol was created to facilitate the patient's decision, as the results of IAF are better that those of DAF. METHODS: We have performed 39 in vitro fertilizations after testicular or epididymal sperm extraction since December 1995. We performed deferred assisted fertilization (DAF) in 19 patients with normal hormone assessment and normal testicular volume or after vasectomy and immediate assisted fertilization (IAF) for 20 patients with an abnormal assessment. RESULTS: The two groups, IAF and DAF, were homogeneous and did not present any differences in terms of age, aetiology of sterility, risk factors or preoperative hormonal parameters. Direct examination of sperm samples, the site of sampling and histological examination did not demonstrate any significant difference between the two groups. ICSI (IntraCytoplasmic Sperm Injection) was performed for 16 couples by IAF and for 14 couples by DAF. We obtained 6 pregnancies (37.5%) in the IAF group and 2 pregnancies (14.3%) in the DAF group. The two groups were identical in terms of the number of oocytes taken and embryos transferred. CONCLUSION: No significant difference was observed between the immediate and deferred fertilization techniques in terms of predictive factors, histology and quality of the direct examination, but the pregnancy rate was higher for IAF. We therefore think that this method should be preferred as the first-line procedure.

Predicting the histologic dating of an endometrial biopsy specimen with the use of Doppler ultrasonography and hormone measurements in patients undergoing spontaneous ovulatory cycles.

OBJECTIVE: To examine the relation between uterine blood flow and endometrial thickness on transvaginal Doppler ultrasonography, serum E2 and progesterone levels, and the histologic dating of an endometrial biopsy specimen obtained in the midluteal phase of a spontaneous cycle. DESIGN: Prospective clinical study. SETTING: A tertiary care infertility center. PATIENT(S): One hundred fifty-nine patients with normal menstrual cycles. INTERVENTION(S): Transvaginal Doppler ultrasonographic evaluation of uterine blood flow and endometrial thickness, determination of serum concentrations of E2 and progesterone, and endometrial biopsy. MAIN OUTCOME MEASURE(S): Resistance index, pulsatility index, serum E2 and progesterone levels, endometrial thickness, and histologic dating of the endometrium. RESULT(S): One hundred thirteen (71%) of the endometrial biopsy specimens showed complete secretory transformation and thus were classified as "in phase," and 46 (29%) of the specimens lacked some or all of the criteria for secretory transformation and thus were classified as "out of phase." There was no statistically significant difference between the in phase and out of phase groups with regard to patient age, endometrial thickness, serum hormone levels, or resistance index. The pulsatility index was significantly higher in the in phase group. The overall predictive value of the studied parameters was only 64% (sensitivity, 57%; specificity, 66%). CONCLUSION(S): Doppler ultrasonographic evaluation of uterine blood flow and measurement of hormone concentrations cannot be used to predict the histologic dating of an endometrial biopsy specimen obtained in the midluteal phase of a spontaneous cycle.

Expression of uteroglobin in the human endometrium.

Uteroglobin is a progesterone binding protein, a member of the antiflammin gene family and possibly a novel cytokine. Initially, uteroglobin was identified as the major protein of rabbit uterine secretion during the phase of preimplantation. Counterparts of the rabbit uteroglobin or its gene are described in rat, mouse, hamster, hare, pig, horse and human. While uteroglobin appears as one of the most extensively studied proteins, particularly its physico-chemical properties, including its crystal structure and its gene, the true physiological role of this protein still remains to be unravelled. Essential to understanding the significance of human uteroglobin in reproductive organs, particularly in the endometrium, is a knowledge of the spatial and chronological expression of this secretory protein. Our studies on 115 volunteers combined reverse transcription-polymerase chain reaction (RT-PCR), immunohistochemistry and quantitative assessment by an enzyme-linked immunosorbent assay for uteroglobin. The expression, localization and release of uteroglobulin in the human endometrium are presented. Secretory uteroglobin is found in endometrial tissue homogenates in highest levels of expression during the early luteal phase (days 15-19, 340 pg/mg total protein). In turn, uteroglobin is released into the uterine lumen in peak amounts during the receptive phase of the menstrual cycle (mid-luteal phase, days 20-23, secretion level 833.4 pg/mg total protein). Our immunohistochemical studies match with these results, as uteroglobin is located during the early and mid-luteal phase in the apical compartments of endometrial gland cells. These observations strongly suggest an involvement of uteroglobin in endometrial preparations for implantation.

Influence of ovarian stimulation on the detection of human papillomavirus DNA in cervical scrapes obtained from patients undergoing assisted reproductive techniques.

OBJECTIVE: To determine whether gonadotropin stimulation influences the detection of human papillomavirus (HPV) DNA in cervical scrapes. DESIGN: Prospective, controlled study. SETTING: Tertiary care infertility clinic. PATIENT(S): Two hundred ninety-four patients enrolled in an IVF or IUI program. Two thousand two hundred sixty-two women from an ongoing screening study who were of similar age served as a control group. INTERVENTION(S): Cervical scrapes were obtained with a cytobrush before and after ovarian stimulation with gonadotropins. MAIN OUTCOME MEASURE(S): Human papillomavirus status was assessed with a general primer (GP) polymerase chain reaction (PCR) using the GP5+/GP6+ system. In GP-PCR-positive samples, high-risk HPV types were identified with a cocktail of digoxigenin-labeled oligonucleotides. Viral load was evaluated by semiquantitative analysis of the PCR products. RESULT(S): The prevalence of high-risk HPVs was 7.8% before stimulation and 6.8% after stimulation and, thus, was similar to the prevalence in controls (8.4%). Twenty-nine patients were positive for high-risk HPVs: 14 were positive before and after stimulation, 6 were negative before and positive after stimulation, and 9 were positive before and negative after stimulation. Positivity for high-risk HPVs and viral load did not correlate directly with serum estrogen levels. CONCLUSION(S): Ovarian stimulation has no significant effect on the prevalence of HPV DNA in cervical scrapes obtained from patients undergoing assisted reproductive techniques.

Chromosomal analysis of multipronuclear zygotes obtained after partial zona dissection of the oocytes.

We have attempted to analyse the chromosome constitution of multipronuclear 1-cell zygotes obtained after partial zona dissection (PZD) of the oocytes. Six cells with three pronuclei could not be evaluated whereas another one was characterized by the presence of a normal haploid and two uninterpretable metaphases. Complete karyotypes were established for 21 tripronuclear cells, taking the varying arrangement of the chromosome sets into consideration. Of the zygotes, 10 showed three separated haploid metaphases (distribution pattern n/n/n), eight zygotes had one haploid and one diploid chromosome set (n/2n) and in three cells the individual sets were not distinguishable (3n). The sex chromosome ratio XXX:XXY:XYY was 7:9:5. Chromosome abnormalities were found in eight of the completely or partially analysable tripronuclear zygotes (36.4%) and included numerical (4 cells), structural (2 cells) as well as combinations of numerical and structural alterations (2 cells). Three out of 11 zygotes with four pronuclei could not be evaluated at all. In three cases, only two chromosome sets were analysable and another cell displayed one uninterpretable set. Three out of eight completely or partially analysable zygotes with four pronuclei (37.5%) had chromosomal abnormalities. Excluding the four cells with one or two uninterpretable metaphases, the sex chromosome distribution XXXX:XXXY:XXYY:XYYY in the zygotes with four pronuclei was 0:1:1:2. Compared with previously analysed multipronuclear zygotes obtained after conventional in-vitro fertilization (IVF), the rate of aberrant zygotes as well as the incidence of aberrant (male + female) chromosome sets were not significantly changed after PZD.

Glass wool filtration leads to a higher percentage of spermatozoa with intact acrosomes: an ultrastructural analysis.

We investigated the possibility of ultrastructural damage to human spermatozoa induced by different sperm preparation techniques. Ejaculates from 20 normozoospermic men were divided into equal aliquots and processed by glass wool filtration, Percoll density gradient centrifugation, and a simple two-step centrifugation procedure which served as a control. The evaluation of 60 spermatozoa from each of 20 test subjects (in all, n = 1200) ensured that a sufficiently large number of spermatozoa were investigated. Ultrastructural damage was assessed by scanning electron microscopy. We investigated the state of the acrosome after sperm preparation and measured the percentage of intact spermatozoal structures compared with that of the control. Compared with Percoll density gradient centrifugation, glass wool filtration yielded a significantly increased proportion of intact acrosomes. However, both preparations gave significantly better results than the control. In conclusion, both glass wool filtration and Percoll centrifugation are efficient techniques for the accumulation of spermatozoa with intact acrosomes. Because of the significantly higher percentage of intact acrosomes, glass wool filtration appears to be the more appropriate method. The significance of the conspicuous bending of sperm tails after Percoll centrifugation is not yet known.

[Obesity--significance in adolescence and for reproduction]. / Adipositas--Bedeutung für Adoleszenz und Reproduktion.

According to the actual knowledge obesity is a serious, nutrition-dependent pathology with a high number of consequences. Endocrine sequence of obesity such as PCO-HAIR-syndrome (polycystic ovarian syndrome, hyperandrogenemia-insulin-resistance) with its cycle disorders and sterility are beginning already in adolescent and women of young reproductive age. With ageing more serious risks such as non-insulin dependent diabetes mellitus (NIDDM), arteriosclerosis followed by coronary disease, stroke and hypertension, metabolic syndrome and a higher prevalence of malignant diseases will appear. Based on these five risks obesity should be treated early when therapeutic strategies are more successful than in older ages. The definition of a diagnosis and the beginning of a weight reduction programme combined with intense motivating treatment as well as medical and psychotherapeutic guidance is an important preventive contribution.

Detrimental effects of polyvinylpyrrolidone on the ultrastructure of spermatozoa (Notulae seminologicae 13).

The effect of in-vitro treatment by polyvinylpyrrolidone (PVP) on the ultrastructure of human spermatozoa has been tested previously with the statistical analysis of B. Baccetti et al. (1995, J. Androl., 16, 356-371). PVP had a primary detrimental action on the plasma membrane, as well as on acrosomal and mitochondrial membranes. Furthermore, membrane damage induces deterioration of the chromatin, axonemal tubules, fibrous sheath, and accessory fibres.

The chromosomal constitution of multipronuclear zygotes resulting from in-vitro fertilization.

We have attempted to analyse the chromosome constitution of 77 multipronuclear uncleaved zygotes obtained from our in-vitro fertilization programme. Complete karyotypes could be established for 51 tripronuclear cells and eight zygotes with four pronuclei. When compiling the results, the varying arrangement of the chromosome sets was taken into consideration. Eighteen tripronuclear zygotes showed three separate haploid metaphases (distribution pattern n/n/n), 16 cells had one haploid and one diploid chromosome set (n/2n), and in 15 zygotes the individual sets were not distinguishable (3n). Two zygotes were in fact tetraploid, the distribution of metaphases on the slide being n/3n and n/n/2n, respectively. In tripronuclear zygotes the sex chromosome ratio XXX:XXY:XYY was 14:16:18, excluding the two tetraploid cells and one zygote with a 23,X/23,X/22,-C or -Y karyotype. Chromosome abnormalities were found in 16 zygotes (31.4%) and included numerical (six cells), structural (four cells) as well as combinations of numerical and structural alterations (six cells). Four of the zygotes with four pronuclei (50%) had numerical and/or structural chromosome aberrations. Excluding two cells with one uninterpretable metaphase and a 22,-C or -Y karyotype, respectively, the sex chromosome distribution XXXX:XXXY:XXYY:XYYY was 1:1:2:1 in zygotes with four pronuclei. Another zygote was found to be pentaploid after fixation. These results suggest that analysis of multipronuclear zygotes yields valuable information about cytogenetic abnormalities occurring at the earliest stage of conception.