S. Milingos

Biological Factors in Culture Media Affecting in Vitro Fertilization, Preimplantation Embryo Development, and Implantation

Abstract: Optimal culture conditions are of paramount importance for in vitro fertilization of gametes, preimplantation embryo development, and implantation for all species. Water is the basis of all culture media, and ultrapure water should be employed. The main energy sources of a medium are lactate, pyruvate, and glucose. The concentrations of the first two vary in different media, whereas the latter is necessary mainly for the later stages (morula to blastocyst) of development. A fixed nitrogen source is essential for implantation embryo development whether this is provided by amino acids, albumin, or serum. Suboptimal culture conditions can block development. Pronuclear zygotes of most species (but not human) arrest at some point between the two‐cell and the 16‐cell stage. Modifying culture conditions can lead the embryos to develop through this block. Hypoxanthine also causes a two‐cell block to mouse pronuclear zygotes, and this again depends largely on culture conditions. Simple culture media are bicarbonate‐buffered systems with pyruvate, lactate, and glucose. Complex media, such as Hams F‐10, contain in addition amino acids and other elements found in serum. Human tubal fluid simulates the fallopian tube microenvironment. EDTA, gonadotropins, growth factors, and other substances can be included in the media to stimulate development. Coculture of embryos with oviductal cells has shown promising results.

Alternative Approaches in the Management of Poor Response in Controlled Ovarian Hyperstimulation (COH)

Abstract: Improving pregnancy rates in patients with many failed attempts remains a challenge during IVF‐ET or ICSI‐ET programs. The availability of good‐quality oocytes is a prerequisite for good results in such programs. The use of a personalized protocol for controlled ovarian hyperstimulation (COH) that gives the best possible results for the specific patient is a main factor for the success in IVF or ICSI. The response of many patients to the ovarian stimulation used is very poor, giving fewer oocytes than expected, resulting in much lower, if any, pregnancy rates. The definition of a poor responder is not clear and differs among researchers. A variety of strategies have been used to improve response in these patients, regardless of the definition used. These include various ovulation induction protocols that we believe might assist these patients achieve a pregnancy. The difficulty is greater due to the fact that poor responders are not a homogeneous group and each patient may have a different cause. More studies with large numbers of patients are needed in order to find those protocols that could provide these couples with an acceptable pregnancy rate.

Metformin administration was associated with a modification of LH, prolactin and insulin secretion dynamics in women with polycystic ovarian syndrome

Aim. To elucidate the dynamics of FSH, LH, prolactin (PRL), TSH and insulin secretion in women with polycystic ovarian syndrome (PCOS) treated with metformin (MET). Patients and methods. In a prospective, controlled and randomised trial, 32 women with PCOS and 32 with normal cycle were recruited to receive MET (850 mg b.i.d.) or placebo (n: 16 for each subgroup) for an average of 40 days. Pituitary function and insulin secretion were assessed before and after intervention by GnRH-TRH tests and oral glucose tolerance test induced insulin response. Results. Basal and area under the response curve (AURC) LH values were higher in PCOS than in normal controls before MET and declined following treatment in the former group (P < 0.05). Ovulatory PCOS responders had lower basal LH, AURCLH and AURCPRL values during MET than anovulatory cases (P < 0.05 for all) and AURCins was lower in ovulatory than anovulatory PCOS before and on MET (P < 0.02–P < 0.05), with a rise of QUICKY index in the former group during MET treatment (P < 0.05). FSH and TSH were similar. Conclusions. MET administration lowered LH activity in all PCOS women and in ovulatory responders and also compromised PRL stimulated secretion in the latter cases. These findings were indicative of an effect of MET on pituitary activity.

Oocyte donation to women over 40 years of age: pregnancy complications

Recently, oocyte donation to women of advanced age has led to a considerable number of conceptions, thus increasing the age limit for becoming pregnant. A main consideration encountered by physicians, though, is the potential medical and obstetric complications of a pregnancy at an advanced age. In this study, the obstetric complications, as well as the perinatal outcome, of pregnancies of aged recipients (above 40) are presented and compared to those of younger recipients. A significantly higher incidence of gestational diabetes (P < 0.001), an increased incidence of pre-eclampsia (at the 10% level of significance) and an increased risk for thrombophlebitis (again at the 10% level) was observed in the older patients, but a careful follow-up during their pregnancy led to a highly satisfactory obstetric and perinatal outcome. A rigorous precycle medical screening (especially for cardiovascular diseases and diabetes) and a careful follow-up during pregnancy is, therefore, imperative so that oocyte donation to older women is not withheld and continues to provide fertility possibilities to otherwise sterile patients.

A preliminary study of the effect of growth hormone on mouse preimplantation embryo development in vitro

The role of growth hormone (GH) in follicular development, ovulation and embryo development is currently under reconsideration. In this study, we have tried to investigate the effect of GH on preimplantation development of mouse embryos in vitro. Zygotes and two-cell mouse embryos were cultured without (control) or with GH. For zygotes, the addition of 0.2 micrograms/ml of GH resulted in 77 +/- 1% of blastocysts formed and 66 +/- 3% rate of hatching (control 64 +/- 4 and 31 +/- 3%, p < 0.05 and p < 0.01, respectively). For two-cell embryos, the addition of 0.2 micrograms/ml of GH resulted in 87 +/- 2% of blastocysts formed and 60 +/- 4% hatching rate (control 76 +/- 4 and 47 +/- 5%, p < 0.05 for both). This positive effect of GH addition implies that the latter can support mouse preimplantation development in vitro and it suggests, along with its local action on the ovary and its possible effects, via the insulin-like growth factor system, on the tubal and uterine epithelium, a continuous role of this hormone in reproductive physiology from follicular maturation to embryonic development and, possibly, implantation.

Factors affecting postoperative pregnancy rate after endoscopic management of large endometriomata

Objective: To identify factors influencing postoperative pregnancy rate in women with extensive endometriosis and large endometriomata as the only identified cause of infertility that were treated by laparoscopy. Method: Sixty‐four infertile patients with endometriomata (≥3 cm) and no other apparent cause of infertility. The latter were removed by operative laparoscopy. Life table calculations, the Students t‐test and the χ2 test were used where appropriate. Result: Thirty‐four patients (53%) became pregnant during the 2‐year follow‐up period. A significantly increased pregnancy rate was found for the first year compared to the second (76 vs. 24%). The existence of adhesions affected adversely the outcome of the operation only as far as early achievement of pregnancy is considered. The number and size of endometriomata and the existence of peritoneal implants have not been found to affect pregnancy rates. The severity of the disease did not affect pregnancy rate, but in the cases with moderate disease most of the pregnancies were achieved during the first postoperative year. The duration of infertility was significantly associated only at the 10% level with decreased pregnancy rates. Conclusion: Extensive endometriosis with large endometriomata can be safely and effectively treated with laparoscopy using the traditional laparoscopic tools providing the infertile patient with a high chance to conceive in a relatively short period of time postoperatively.

The in vitro development of mouse embryos beyond the blastocyst stage into the hatching and outgrowth stage using different energy sources

AbstractPurpose: The purpose of this study was to investigate the effect of male and female serum supplementation on the in vitro development of mouse embryos beyond the blastocyst stage until the outgrowth stage since the latter may be related to the nidation of the embryo. We also studied the effect of EGF addition on embryo culture and blastocyst outgrowth. Methods and Results: The blastocyst and hatching rates of two-cell mouse embryos cultured in Hams F-10+BSA, Hams F-10+male serum, or Hams F-10+female serum were found to be comparable (P>0.05). The outgrowth rate of hatched blastocysts was significantly increased, though, when they were transferred to 50% male serum compared to either 50% BSA or 50% female serum (P<0.01 and P<0.05, respectively). In the last experiment, either 100 or 150 ng/ml EGF was added to the culture medium from the two-cell stage till blastocyst development and the latter were cultured till outgrowth in 50% BSA, male serum, or female serum. For both concentrations of EGF, the outgrowth rate was significantly higher in male serum compared to the other conditions (P<0.01 and P<0.05, respectively). The outgrowth rate was also higher when EGF was used compared to plain medium before transferring the blastocysts to either male or female serum (P<0.01 for both). Conclusions: We conclude that the development of embryos to the outgrowth stage is significantly enhanced by male serum. The addition of EGF from the two-cell stage also significantly improves the outgrowth success rate for both male and female serum conditions.

Knowledge attitude and behavior of female students concerning contraception in Athens Greece.

The aims of this study were to investigate the knowledge, attitudes and behavior of female high school and university students concerning contraception. The study was performed at the Alexandra University Hospital in Athens. A total of 297 female students participated in the study. A questionnaire was used to evaluate the depth of knowledge and attitude of respondents concerning contraception. Our findings suggest students have a defective knowledge of contraception. Their main source of information were friends but the majority would prefer to receive information from doctors. The most popular contraceptive method was the male condom. The attitude and behavior of young women in our study were consequently defective. In conclusion, there is a need to provide students with correct, detailed and broad-based information on conception and contraception as part of the school curriculum to help them acquire adequate knowledge and develop appropriate attitudes on contraception and sexual health.

Does pre-treatment with micronized progesterone affect the ovarian response to a gonadotropin releasing hormone agonist flare-up protocol?

The purpose of this study was to investigate the ovarian response and the receptivity of the endometrium in women pre-treated with micronized progesterone. Eighty-two normogonodotropic women undergoing in vitro fertilization were studied. Thirty received micronized progesterone 1500 mg/day from day 21 of the cycle for a minimum of 2 weeks ,and 52 did not receive micronized progesterone (control group). A gonadotropin releasing hormone agonist (GnRH-a) was administered to all the patients in the follicular phase (flare-up). Twenty-five cycles were cancelled for fertilization failure due to male factor, 12 (40%) in the progesterone group and 13 (25%) in the control group (p = 0.271). There was no difference in the number of oocytes retrieved (7.3 ± 5 vs. 8.2 ± 4) ,fertilization rate (50.8% vs. 65%) ,clinical pregnancy rate (16.6% vs. 25%) or implantation rate (8% vs. 14%). In the progesterone group cases without fertilization ,we performed two biopsies to evaluate the receptivity of the endometrium. Pinopode expression was noted 7 days after oocyte retrieval. It seems that the administration of micronized progesterone in the previous cycle does not affect the ovarian response to the combination of follicular phase GnRH-a and gonadotropins ,nor the receptivity of the endometrium.

Effect of human hydrosalpinx fluid on the development of mouse embryos and role of the concentration of growth factors in culture medium with and without hydrosalpinx fluid

The aim of this study was to investigate the effect of human hydrosalpinx fluid (HF) on the development and blastulation of mouse embryos and the role of the concentration of growth factors in culture medium with and without HF. In total, 2100 mouse embryos were cultured. Female mice were induced to superovulate and then mated with males. Two-cell-stage embryos were recovered from the oviduct and cultured in Hams F-10 medium with bovine serum albumin and HF. Epidermal growth factor (EGF) and insulin-like growth factor-I (IGF-I) were analyzed by quantitative enzyme immunoassay. Mean blastulation index of 1.11, 0.97 and 0.98 was found at HF concentration of 5%, 20% and 30%, respectively (p = 0.8). The mean value of EGF in the control culture medium without HF was 11.2 pg/ml, which was statistically significantly different from that in culture medium containing HF (p < 0.001). The mean value of IGF-I in the control group without HF was 1.30 pg/ml and was not statistically significantly different from that in culture medium containing HF. Development of the two-cell-stage embryos was not affected at low (< 30%) HF concentrations. In conclusion, the present study demonstrates that even apparently normal blastulation is affected by any concentration of HF because of low embryonic EGF.