Since the first successful human pregnancy from in-vitro fertilization (IVF) was achieved (Steptoe and Edwards, 1978), assisted reproductive technology has become the frontier of birth infertility treatment and research. There have been continuous improvements in the pregnancy and birth rates with IVF.

These improvements have been directly attributed to advances in the hormonal stimulation of patients with various controlled ovarian hyperstimulation (COH) protocols and improved culture media and culture systems for oocytes, sperm, and embryos. However, through all these improvements with stimulated cycles, there has been continued development with natural, unstimulated, or limited-stimulation cycles followed by in vitro maturation (IVM) of oocytes. Any protocol that would decrease the amount and duration of hormonal stimulation before oocyte retrieval would have an advantage over the more common COH/IVF protocols if the resulting pregnancy rates were the same or improved (Cha K. et al, 1991). Research in in-vitro maturation (IVM) of the human oocyte has shown significant progress and provided hope for certain groups of patients who have either poor response or have a high risk of developing ovarian hyperstimulation. Human oocytes recovered from immature follicles, following retrieval can resume and complete meiosis in-vitro when cultured in media supplemented with recombinant follicle Stimulating Hormone (r-FSH) and Human Chorionic Gonadotrophin (hCG) (Trounson A., et al, 2001; Hreinsson J. et al, 2003; Lin Y. et al, 2003).

Several reports showed that in-vitro matured oocytes could be fertilized, and result in pregnancy (Jaroudi K., et al, 1999), and birth of healthy babies (Chian R., et al, 2001, Suikkari A. et al, 2005). Despite the clinical utilization of IVM in the field of human reproduction, its pregnancy and birth rates remain low compared to in-vivo matured oocytes (Lui J., et al, 2003, Lin Y.,et al, 2003).

This study aims to study the effect of the follicles' size on oocyte maturation, fertilization and cleavage, and to compare the outcomes of in-vitro maturation of immature oocytes recovered in situ from infertile women with polycystic ovaries, versus poor responders.

• To determine the effect of recombinant follicle stimulating hormone (r-FSH) concentration in culture media on in vitro maturation outcomes.
  
• To determine the effect of the follicle size on in vitro maturation outcomes.
  
• To determine the in vitro maturation outcomes of immature oocytes recovered from infertile patients with polycystic ovaries (PCO) and poor responders.

This study was conducted during the period from April 2000 to December 2004. The results of this study were collected and evaluated from the Assisted Reproduction Unit at Almana General Hospital, Dammam, Eastern Province of the Kingdom of Saudi Arabia and incorporation with the department of Obstetrics and Gynecology, Campus Benjamin Franklin, Charité, Homboldt University. The study was approved by the research ethics board of the hospital. A written informed consent was obtained from all patients. To decide which concentration to use in the culture medium, a preliminary experiment was performed to compare 2 concentrations (7.5 IU/ml and 0.075 IU/ml) and compared to a medium with no FSH.

(A) The effect of r-FSH concentration in the culture media on in-vitro maturation of oocytes

Recombinant FSH in the culture media was used in two concentrations of 0.075, 7.5 IU/ml and none as a control.

This experiment was performed on patients producing more than 15 germinal vesicles (GV) oocytes. Immediately after collection, the oocytes were equally distributed in three groups of 5 oocytes each in three center-well dishes containing 3ml Ham's F10 media.

Two r-FSH concentrations of 0.075, 7.5 IU/ml were added in two dishes and none as control, respectively. Oocytes maturation was evaluated 30 hours after incubation. Oocytes maturation, fertilization, cleavage and pregnancy rates were assessed.

(B) The effect of follicular size on the rate of oocyte maturation

In this experiment, oocytes were collected from 50 PCO patients (Group 2) who were stimulated with a daily dose of 300 IU (Purgeon, Organon, Holland) for 5 days, starting on day 2 of the menstrual cycle until day 6 when a transvaginal ultrasound scan was performed. The scan showed follicular size ranging between 8-13 mm. The PCO patients were divided into two subgroups according to follicular size on the day of hCG injection; Group 2I had a follicular size of 8-10 mm and Group 2II had a size of 11-13 mm. The oocytes retrieved from these patients were incubated for 30 hours in Ham's F10 supplemented with 0.075 IU r-FSH/ml. Oocyte maturation, fertilization, cleavage, and pregnancy rates were then assessed.

(C) In vitro maturation outcomes of immature oocytes recovered in-situ from infertile patients with polycystic ovaries (PCO) and poor responders

This part of the study was conducted on 40 infertile women (Group 3). This group was further divided into two subgroups, 20 infertile women with polycystic ovaries (Group 3I) and 20 poor responder infertile women (Group 3II). The timing of the start of treatment was random, as most of the patients had an irregular menstrual cycle.

Infertile women with polycystic ovaries and poor responders were included in this study during their schedule for ICSI in the IVF program. The patients were recruited at random. It was fully explained to each patient that the procedures related to the study were not part of the routine diagnostic procedures required for their infertility assessment.

Patients were recruited after PCO was identified by:

1. Pelvic ultrasound (the ultrasonic criteria of PCO were essential for the diagnosis. It included the presence of more than 8 small follicles of 2-8 mm in diameter around a dense core of stroma and a dense ovarian capsule),
   
2. Their endocrine and clinical features varied between regular and irregular cycles,
   
3. Anovulatory polycystic ovarian syndrome patients had characteristically elevated androgen level, LH: FSH ratio >2, and frequently, the clinical features of hirsutism and increased body weight (Adams J. et al., 1985; Hershlag A. et al, 1996).

Poor responders are patients who fulfilled one or more of the following criteria:

1. Failed to achieve estradiol concentration above the level of 200 pg /ml on the day of hCG (Garcia J. et al, 1983).
   
2. Produced less than three mature follicles during the previous stimulation attempts (Serafini P. et al, 1988).
   
3. Failure and/or cancellation of previous IVF cycles due to low quality of oocytes retrieved in previous stimulations (Rienzi L. et al, 2002).

Oocytes retrieval and IVM Procedure
Transvaginal ultrasound guided oocytes collection was performed using a specially designed 17-G single-lumen aspiration needle (Casmed, UK) with a reduced aspiration pressure of 7.5 kpa. Aspiration of the follicles was performed under general anesthesia for all patients. All patients received an antibiotic cover of a single dose of 500 mg of metronidazole intravenously during the procedure.

Oocytes were collected in culture tubes containing warm Earl's balanced salt solution with 5000 IU/ml heparin. Immature oocytes were incubated in a culture dish containing 1ml of 3M (Medicult) medium supplemented with r-FSH (Puregon, Organon) (according to the stage of the study) and 5.00 IU/ml hCG (Pregnyl, Organon) at a temperature of 37°C in an atmosphere of 5% CO2 and 95% air with high humidity. After culture, the maturity of the oocytes was determined under the stereomicroscope at 30 hours post collection. Oocytes were denuded of cumulus and maturity was determined by the presence of the first polar body. Suitable oocytes were injected with single spermatozoa by micromanipulation (Research Instrument, UK). Following ICSI, each oocyte was transferred into 1ml of Medi-cult IVF medium in a tissue culture dish. Fertilization was assessed 18 hours after ICSI for the appearance of two distinct pronuclei and two polar bodies. Oocytes with two pronuclei were further cultured in Medi-cult IVF medium. Embryos were transferred on day 2 or 3 after ICSI.

Statistical analysis was done by the student's t - test. Frequency data were analyzed by ?2 contingency tests. Embryo development ratio data were analyzed by analysis of variance. Values were considered significant when P<0.05. Since the oocytes were not matured and inseminated at the same time following maturation in culture; the development stages of embryos were variable both within and between patients.

The results of the present study were based on data generated from the two experiments. The mean duration of infertility was 12.3 ± 4.6 years for all patients of the study groups. All patients were under 45 years of age with a range of 21 - 44 years (mean 35.1 ± 5.3 years).

Results of the First experiment (FSH Concentration)
The first experiment was designed to define the optimum r-FSH concentration. Our data showed that 0.075 IU/ml was the optimum concentration that provided higher maturation, fertilization and pregnancy rates compared to 7.5 IU/ml and the control. Details regarding the number of oocytes collected, maturation, fertilization and pregnancy rates after in vitro maturation in media containing different concentrations of r-FSH are shown in table 1. Recombinant FSH concentration had significantly (p<0.05) increased the rate of oocyte maturation from 47% at 0 concentration to 81% and 83% at 0.075 and 7.5 IU/ml, respectively. Fertilization, cleavage, and clinical pregnancy rates showed a similar trend and significantly increased from 45% to 83% and 80%; from 32% to 80% and 77% and from 0% to 17% and 14% at the three concentrations, respectively. The results however showed that increasing r-FSH concentration to levels more than 0.075 IU did not further improve maturation, fertilization, cleavage and pregnancy rates even when the concentration was increased up to 100 folds. Our data showed that 0.075 IU/ml was the optimum concentration that provided higher maturation, fertilization and pregnancy rates compared to 7.5 IU/ml and the control.

Results of the Second experiment (Follicular Size)
Based on the optimum rFSH concentration as decided from the first experiment, the second experiment was designed to study the effect of the follicle size on oocytes maturation, fertilization and developmental competence.

The concentration of rFSH chosen was 0.075 IU.

The results of two different follicle sizes (group 2) are shown in table 2. Oocytes retrieved from 11-13 mm follicles showed higher rates of maturation, fertilization and pregnancy, than those retrieved from 8-10 mm follicles. The above parameters increased from 48 to 70%; from 54 to 76% and from 11 to 22.5%, in the two follicle sizes, respectively. In the first subgroup study (group 2I), the 6 and 5 pregnancies resulting from oocyte cultured in media containing 0.075 and 7.5 IU/ml all ended in delivery of healthy children. On the other hand, in the second subgroup (group 2II), the two pregnancies resulting from 8-10 mm follicles size completed full term, whereas two of the nine pregnancies in the 10-12 mm follicle size subgroup ended in miscarriage and the remaining seven pregnancies ended in delivery of healthy babies. Follicular size showed significant (P<0.05) effect on the assessment parameters.

Results of the Third experiment (PCO and Poor responders)
The mean age of group 3 patients was 32.3 ± 5.8 for PCO patients and 36.4 ± 7.1 for the group of poor responders. The means of parity, abortion, and Hb% were also comparable between the two sub groups. Both the body mass index (29.7 ± 2.3 vs. 27.1 ± 1.6) and the duration of the cycles (53.2 ± 21.3 vs. 30.3 ± 8.6) were higher in group 3I (table 3). There was however no difference between the two subgroups (group 3I and group 3II) in the concentrations of estradiol, progesterone, FSH, and LH on day 2 and on the day of oocytes retrieval (tables 2).

From 12 irregular PCO women (G3Ia), the mean numbers of oocytes recovered, matured in vitro, and fertilized after insemination, and cleaved in culture were 18.1, 11.7, 4.2, and 3.1, respectively. The mean numbers of oocytes recovered, matured, fertilized and cleaved from 8 regular cycling PCO women (G3Ib) were 5.4, 4.4, 3.1 and 1.6, respectively. Oocytes recovered from regularly cycling patients had a higher developmental potential when compared with irregular and anovulatory patients with significantly (P < 0.05) including higher maturation and fertilization rates (table 4). Cleavage was not significantly different between the two subgroups, although there was a trend to increased cleavage of embryos in the regular cycling group. Moreover, embryos produced from regularly cycling patients had a significantly higher embryo development ratio (P< 0.05), indicating the faster cleavage rate of embryos produced from this group of patients. Embryo development ratio is defined as the observed cleavage stage/ the expected cleavage stage × 100. Three pregnancies were obtained, one has delivered a pre-term at 36 weeks and two miscarried at 8 and 10 weeks.

From 20 poor responder women (Group 3II), the mean numbers of oocytes recovered, matured in vitro, fertilized after insemination, and cleaved in culture were 18.1, 14.5, 5.1 and 3.4 respectively (table 5). The embryo development ratio was 63.4 ± 2.6. One pregnancy was obtained with the delivery of a full term female baby.

The feasibility of obtaining full-term pregnancies from in vitro-matured immature oocytes obtained from stimulated and non-stimulated ovaries is well established (Veek L. et al, 1983; Cha K. et al, 1991). The scarcity of subsequent reports points to the fact that the procedure is not even close to being transferred into daily clinical work. The impossibility of judging ooplasmic maturation forces the use of nuclear maturation as the basis for classification of female gametes.

The present study showed that in-vitro matured oocytes retrieved from PCOS patients had the potential to undergo successful maturation, fertilization and the resultant embryos and showed good developmental competence. Following the IVM procedure, embryo transfer culminated in clinical pregnancies and birth of healthy children. All the oocytes retrieved in this study were at the germinal vesicle (GV) stage. The latter is defined as the stage that represents oocytes arrested at prophase of meiosis-1 with prominent discernable germinal vesicle nucleus.

There are various factors that affect oocyte in-vitro maturation. The most important among these factors are: the exposure of the immature oocyte to gonadotrophins in the culture media, and the follicle size at which the oocyte was retrieved. r-FSH, LH, and hCG (Hreinsson J. et al, 2003), and purified gonadotrophins (Mikkelser A.L.et al, 2001) were used to induce oocyte maturation in vitro. In this study the effect of both factors on oocyte maturation, fertilization, cleavage, and pregnancy rates were investigated.

Cha et al, reported a pregnancy rate of 27.1% after IVM and IVF-ET in patients with PCOS. They also reported that the combined ET (ZIFT + uterine ET) yielded a significantly higher pregnancy rate than either ZIFT alone or uterine ET alone (Cha K., et al, 2000). Previously, other studies have shown that the pregnancy rate of conventional IVF in PCOS patients was similar to that of conventional IVF in non-PCOS (MacDougall M., et al, 1993). A possible mechanism suggested for the lower pregnancy rate of IVM is that some of the oocytes undergoing nuclear maturation after IVM are incapable of undergoing cytoplasmic maturation, thus resulting in poor embryo quality and a higher incidence of pregnancy failure. A number of other factors might lead to a lower success rate of IVM, including sub-optimal culture conditions, advancing maternal age, an endocrine disturbance, previous IVF failures, and sub-optimal timing of insemination (Picton H., 2002).

Table 6: Compares different oocyte in-vitro maturation rates obtained from various studies to the rate obtained in our study.

The variations in maturation rates between the present study and those mentioned in table 6 may be due to the composition of the culture medium used and protein supplement. In the present study our optimum culture time (30h) was comparable with other studies (Cha K.Y. et al, 1998). Inadequacies of cultured media could not be ruled out as a possible cause for low IVM success (Combelles C.M. et al, 2002). There is evidence suggesting that culture media used for IVM adequately support nuclear maturation, but failed to produce oocyte with cytoplasmic maturation. While we used synthetic serum supplement as a sources of proteins, in other studies fetal bovine serum (FBS) was used. FBS was considered more crucial for bovine oocyte maturation than human. In addition, our base medium used was Hams F10, which is designed to meet the nutritional and maturational needs of human oocyte.

On the other hand the effect of follicle size on the above parameters showed some interesting results. Our data showed that oocyte maturation rate and developmental competence has significantly increased with increase in follicle size. Eppig J.J. et al, (1992), concluded that developmental competence of oocytes depends on the follicle and oocyte size. The growth in size is due to the fact that oocyte synthesizes and stores mRNA and proteins that are essential for the completion of maturation and for the subsequent acquisition of embryo developmental competence (Gosden R. et al, 1995). This probably explains the relatively higher maturation, fertilization, cleavage, and pregnancy rate in oocyte obtained from follicle with 11-13 mm size rather than 8-10 mm. While in the present study pregnancy rate increased with follicle size, other reports found that implantation, pregnancy, and birth rates were independent of follicular size (Wittmaack F. et al, 1994; Salha O. et al, 1998). Our data suggested that when oocytes were cultured in IVM media containing 0.075 IU r-FSH or retrieved from follicle size exceeding 10mm, a comparable or even better maturation rates and developmental competence than results shown in several recent reports were obtained.

Also, the results indicate that in vitro maturation of oocytes recovered from PCO patients exhibit developmental competence more than the oocytes recovered from poor responder patients. The explanation of this finding might be related to the age of the patients where most of the poor responders are older than PCO patients with the contribution of other factors like the male factor, whereas PCO patients are younger and their main problem was the competency of in vivo oocytes maturation. In studying the effect of the male factor, all males in PCO patients had normal sperm count and viability. In poor responders, only 1 case showed subnormal sperm count. The effect of the male factor could be omitted in our study due to the low number of cases and the inability of any statistical evaluation.

IVM is a useful treatment option for women with PCOS or who are resistant to FSH with less risk of Ovarian Hyperstimulation. As this group of patients are resistant to gonadotropin stimulation for various reasons and as they require prolonged and higher doses of gonadotrophin stimulation protocols, IVM provides a different approach to a safer and cheaper treatment modality. In addition, natural-cycle IVF combined with IVM might provide more efficient treatment for poor responder infertile women.. Oocyte maturation and embryo development are less in women with irregular menstrual cycles.

In the present study factors affecting immature oocyte maturation and developmental competence are not fully explored. There are many gaps that need to be bridged and other factors need to be closely investigated. The effect of growth hormone in culture media during oocyte maturation, chromosomal anomalies as well as the effect of anesthesia is a worthwhile thorough investigation.

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