Follicular fluid protein content (FSH, LH, PG4, E2 and AMH) and polar body aneuploidy

Abstract

Purpose

Our objective was to identify a marker for oocyte aneuploidy in follicular fluid (FF) in women with an increased risk of oocyte aneuploidy after controlled ovarian hyperstimulation.

Materials and methods

Three groups of oocytes were constituted for polar body screening by FISH (chromosomes 13, 16, 18, 21 and 22): Group 1, advanced maternal age (n = 156); Group 2, implantation failure (i.e. no pregnancy after the transfer of more than 10 embryos; n = 101) and Group 3, implantation failure and advanced maternal age (n = 56). FSH and other proteins were assayed in the corresponding FF samples.

Results

Of the 313 oocytes assessed, 35.78 % were abnormal. We found a significant difference between the follicular FSH levels in normal oocytes and abnormal oocytes (4.85 ± 1.75 IU/L vs. 5.41 ± 2.47 IU/L, respectively; p = 0.021). We found that the greater the number of chromosomal abnormalities per oocyte (between 0 and 3), the higher the follicular FSH level.

Conclusion

High FF FSH levels were associated with oocyte aneuploidy in women having undergone controlled ovarian hyperstimulation.

Introduction

In humans, 50 % of fertilized embryos do not implant [31] and most of these implantation failures are related to aneuploidy. Moreover, these abnormalities are predominantly oocyte-related, since between 20 % and 30 % of human oocytes have defects of this type [48]. When screened with respect to 10 chromosomes, only 36 % of embryos were found to be normal after ovarian hyperstimulation for IVF [3]. However, it is not clear why our species has such a high chromosomal risk.

Meiosis I errors can occur via one of two mechanisms: (a) reduced cohesion between sister chromatids, leading to premature separation of sister chromatids (PSSC) and an abnormality affecting a single chromatid (many authors consider that this is the major event in oocytes from older women [21, 44, 45, 66, 69]), and (b) a malsegregation of a whole chromosome (comprising 2 chromatids at this stage) due to a change in the spindle structure, i.e. non-disjunction (ND).

Over the last 15 years or so, it has been unambiguously demonstrated that oocyte chromosomal abnormalities are strongly related to maternal age [5, 15, 22, 25, 44–46]. Many reports on the FISH-based preconception screening (PCS) of 5 chromosomes have revealed a first polar body (PB) abnormality rate of between 22 % and 42 % in women over the age of 36 [65], 1998, [30, 41, 52, 66, 67, 69]. Besides age, factors like hyperstimulation treatment may also influence the results. The effect of superovulation and the relative importance of meiosis I and II in the generation of aneuploidy are still subject to debate in the literature. Although most errors in natural human reproduction were thought to occur during meiosis I (though with some dependence on the chromosome involved) [23, 24], recent comparative genomic hybridization (CGH)/microarray data on first and second polar bodies after IVF prompted different conclusions [15, 33]. Pre-IVF ovarian stimulation could perturb meiosis in aging oocytes and also modify the aetiology of meiotic errors [15]. Baart’s randomized, prospective study of an embryo screening programme [4] showed that the embryonic aneuploidy rate was significantly higher in women undergoing a conventional, high-dose FSH hyperstimulation protocol than in women undergoing “mild” (i.e. low-dose) hyperstimulation. An impact of ovarian stimulation on aneuploidy has also been suggested by many authors [4, 12, 32, 40], although other groups have reported contradictory results [10, 36, 68]. In a study of human in vitro matured oocytes, the error rate for the first meiotic division increased significantly [75] with increasing FSH levels in the culture medium. Lastly, the results of an European collaborative study on first and second PB screening in aged women [21] were compatible with the hypothesis in which aneuploidies are more frequent in aged women undergoing IVF than in natural conception of same age women. The study also suggested that the type of aneuploidy differs in these two situations since in this series, over half of aneuploidies resulted from errors in the second meiotic division. More generally in humans, many lines of evidence argue in favour of interactions between the hormonal environment (especially FSH levels), the quality of oocyte maturation, the quality of meiosis and the risk of aneuploidy. Kan [28] found a positive correlation between perifollicular vascularisation during ovarian stimulation, normal chromosome content in the oocyte and the embryo implantation rate. Perifollicular hypoxia may have a harmful impact on the configuration of the meiotic spindle and on embryo quality [62, 63]. Smoking is thought to dampen the response to ovarian stimulation and increase the oocyte aneuploidy rate [76]. Recent work has underlined the importance of factors in the ovarian environment (such as the level of dehydroepiandrosterone) [19] and the oocyte’s micro-environment (i.e. the cells in the cumulus-oocyte complex) on oocyte quality [1, 2, 17].

Results obtained in mice also suggest that subtle changes in the regulation of folliculogenesis and oocyte maturation are capable of inducing aneuploidy [49, 50]. have shown that FSH modifies the spindle configuration. Hence, exposure to high doses of FSH during in vitro maturation could accelerate nuclear maturation and increase the aneuploidy rate in metaphase II. An elevated oocyte aneuploidy rate has been observed in an LH gene knock-out mouse [26]. Furthermore, [61] showed that repeated FSH stimulation can alter meiotic spindle quality in the mouse. Lastly, aging-associated aneuploidy in mice can be prevented by caloric restriction [51].

Hence, the objective of the present study was to identify follicular fluid (FF) markers that are predictive of the risk of aneuploidy, in order to gain insight into the mechanism involved in various groups of women with an increased aneuploidy risk.

In addition to older women, a number of other subpopulations present an elevated oocyte aneuploidy rate. This includes women with implantation failure (i.e. the lack of a pregnancy after the transfer of at least 10 embryos) [53, 70]. However, non-invasive, early screening of these abnormalities is not yet possible.

We evaluated the predictive potential of several factors involved in oogenesis and ovulation: FSH, LH, P4, E2, testosterone and anti-Müllerian hormone (AMH). Plasma AMH may be a marker of ovarian function and the likelihood of pregnancy in Assisted Reproductive Technology (ART) programmes [11, 27, 34, 47] and thus also appears to be a good candidate marker of oocyte quality.

Most of the previous studies of FF proteins involved pooled samples, which prevented an individual analysis of each follicle obtained after ovarian stimulation and the associated outcome for each oocyte. Our study is novel in that we tracked individual oocytes and corresponding FF samples.

Materials and methods

The study population

We recruited all the women with normal karyotype participating in our intracytoplasmic sperm injection (ICSI) programme with a PCS protocol for age or implantation failure at the Poissy Saint-Germain-en-Laye ART Centre. First PB diagnosis is performed before fertilization in patients who have a normal karyotype but present known risk factors for oocyte aneuploidy and not in a control group without any aneuploidy risk factor due to the restrictions of french bioethics law. In France, these couples with normal karyotypes though at risk for oocyte aneuploidy can not undergo pre-implantation genetic diagnosis (PGD) because the latter is not authorized when the parents are free of genetic abnormalities (even in cases of advanced maternal age and repeated implantation failure). Seventy-eight oocytes retrievals for FISH PCS test were performed on the first PB, with the individual collection of FF samples. Three groups were constituted, as a function of the indication:

1. (advanced maternal age): 28 patients, 40 ICSI with PCS attempts (156 oocytes with a PB diagnosis) in patients over the age of 36 (mean ± SD age: 38 years and 11 months ± 1 year and 11 months) but below the threshold for implantation failure indication (i.e. fewer than 10 embryo transferred prior to the attempt in question). 32/40 attempts (80 %) were ICSI for severe male infertility.

2. (implantation failure): 17 patients, 20 ICSI with PCS attempts (101 oocytes with a PB diagnosis) in patients under the age of 36 (mean ± SD age: 32 years and 1 month ± 2 years and 7 months) and with implantation failure (i.e. more than 10 embryos transferred (mean: 14.5 ± 3.6) and no pregnancy prior to the attempt in question). 12/20 attempts (60 %) were ICSI for severe male infertility.

3. (implantation failure + advanced maternal age): 15 patients, 18 ICSI with PCS attempts (56 oocytes with a PB diagnosis) in women over the age of 36 (mean ± SD age: 38 years and 3 months ± 1 year and 7 months) with implantation failure (13 ± 3 embryos transferred and no pregnancy prior to the concerned attempt). 13/18 attempts (72 %) were ICSI for severe male infertility.

Methods

Ovarian stimulation and FF collection

Follicular development was stimulated with recombinant FSH (Gonal F® from Merck serono orPuregon® from MSD) and was monitored by E2 mesurements and transvaginal ultrasound from day 5 after stimulation. Ovulation was induced with 250 μg of recombinant hCG (ovitrelle® from Merck serono). Oocyte puncture and FF sampling were performed on individual follicles. The FF’s volume and aspect (haemorrhagic, haematic, orange or yellow) were noted. Ten samples (3 %) were excluded because the blood contamination level was greater than 3 % (corresponding to a haemorrhagic aspect). The non-excluded FF samples (with a haematic, orange or yellow aspect) were divided into 250 μl aliquots and frozen at −80 °C for subsequent analysis.

Assay for FF markers

Assays for some putative FF markers (FSH and LH) were performed on an ARCHITECT system in Two-step, using an immunoassay microparticulate by chemoluminescence (CMIA) (Abbott, Rungis, France).

Oestradiol, progesterone and testosterone were performed on an ARCHITECT system in one step, using an immunoassay microparticulate by chemoluminescence (CMIA) (Abbott, Rungis, France).

Anti-Müllerian hormone was assayed quantitatively with the AMH/MIS ELISA kit (Immunotech, Marseilles, France).

Total protein was assayed on a COBAS INTEGRA 800 system, using a Colorimetric test (Roche, USA).

FISH PCS test

Polar body biopsy was carried out as previously described [69] using the Zilos TK laser (Hamilton Thorne Biosciences, Boston USA). Immediately after oocyte retrieval and corona radiata removal, 3 or 4 laser impacts (180 mW, 0.5 ms pulse) were made in the zona pellucida. The PB was then extracted (from 340 oocytes, in all) with a biopsy micropipette (Humagen, Charlottesville, VA, USA) and placed in 0.5 μl of water on a siliconized slide. The drop was air-dried and two drops of Carnoy solution were added.

The slide was transferred into a series of solutions: methanol at room temperature for 5 min, 2× SSC at 37 °C for 10 min, 1 % paraformaldehyde at room temperature for 10 min, PBS at room temperature for 5 min, 0.1 N HCl with 30 μl/40 ml of 10 % active pepsin solution for 10 min, PBS at room temperature for 5 min and, lastly, 70 %, 85 % and then 100 % ethanol solutions for 2 min each.

A 3 μl drop of probe solution was placed on each PB. Codenaturation and hybridization were automatically performed in a Hybrite system (Vysis) (at 73 °C for 4 min and at 37 °C for 4 h).

The slide was then washed in a 0.7× SSC/0.3 % NP40 solution for 1 min 45 s and then in a 2× SSC/0.1 % NP40 solution for 15 s. This was followed by counterstaining with an antifade solution and analysis with a five-filter fluorescence microscope (Olympus BX60) and the PathVysion imaging system.

Polar bodies were analysed using the MultiVysionTM Polar Body Kit probe panel hybridization mixture (Abbott), which includes LSI® 13 (13q14, labelled with SpectrumRedTM), CEP® 16 (satellite II D16Z3, labelled with SpectrumAquaTM), CEP® 18 (alpha satellite D18Z1, labelled with SpectrumBlueTM), LSI® 21 (21q22.13–21q2.2, labelled with SpectrumGreenTM) and LSI® 22 (22q11.2, labelled with SpectrumGoldTM). Chromosomes 13, 16, 18, 21 and 22 were chosen because those aneuploïdies are the most viable autosome aneuploïdies in the human species.

Each first PB chromosome normally consists of two chromatids. Locus-specific probes always give a doublet signal corresponding to each of the two chromatids; hence, each chromosome was represented by either a doublet of a distinct colour (sometimes very close) or by two separate signals. Centromeric probes usually give strong signals for one chromosome or a doublet with very close signals. The absence of spot (0 spot; no chromosome present in polar body) or the presence of 4 signals (2 chromosomes present in polar body) was interpreted as ND and 1 or 3 spots were interpreted as PSSC. Two separate signals were considered to correspond to balanced separation of sister chromatids (rather than an abnormality) and were therefore not included in the PSSC count.

313/340 oocytes could be successfully diagnosed and we had 27 technical failures

The ICSI procedure

We decided in the 3 groups to inject all oocytes with normal FISH results or lacking a FISH analysis. Indeed some oocytes were immature in the morning at the time of the biopsy but expelled their polar body a few hours later and could be injected in the afternoon. The reason to do so was to inject as many oocytes as possible to preserve all the possibilities of pregnancies since the efficiency of the procedure in terms of pregnancy rates has not yet been demonstrated. The opening in the zona pellucida was maintained in a 12 or 6 o’clock position, in order to keep the spindle away from the microinjection site. Oocytes were then microinjected using a 7 μm outer diameter ICSI micropipette (Humagen).

Statistical analysis

Statistical analyses (chi-squared, Fisher, ANOVA and t tests) in Tables 1, 2, 3 and 4, and ANOVA and t test in Figs. 1, 2 and 3, were performed using Statview software. The threshold for statistical significance was set to p < 0.05.

Table 1.

Patients characteristics in the 3 study subgroups and stimulation data

	Group 1 : Advanced maternalage	Group 2 : Implantation faillure	Group 3 : Implantation faillure + advanced maternal age	p value
Number of patients with severe male infertility (%)	25/28 (89 %)	12/17 (70.5 %)	11/15 (73 %)	NS
Number of attempts with severe male infertility (%)	32/40 (80 %)	12/20 (60 %)	13/18 (72 %)	NS
Age (mean ± SD)	38 years and 11 months ± 1 year and 11 months	32 years and 1 months ± 2 year and 7 months	38 years and 3 months ± 1 year and 7 months	1 vs 2 and 2 vs 3: p < 0.0001
				1 vs 3: NS
FSH units administered per attempt (mean ± SD)	2237.86 ± 624.94	2836.83 ± 2827.50	2805.00 ± 1011.73	1 vs 2: p = 0.0004
				1 vs 3:p = 0.006
				2 vs 3: NS
E2 serum level 36 hours before oocytes retrieval (mean ± SD)	2013.47 ± 688.06	2299.82 ± 1049.85	1592.85 ± 689.50	1 vs 2: p = 0.0004
				1 vs 3 and 2 vs 3:p < 0.0001
Number of oocytes retrieval (mean ± SD)	8.27 ± 3.19	9.25 ± 2.54	6.29 ± 2.27	1 vs 2:p = 0.0005
				1 vs 3 and 2 vs 3:p < 0.0001
Day 3 basal FSH (mean ± SD)	6.79 ± 0.68	6.43 ± 3.49	9.94 ± 6.00	1 vs 2: NS
				1 vs 3 and 2 vs 3:p = 0.003
Day 3 LH (mean ± SD)	5.72 ± 2.86	4.53 ± 1.35	4.20 ± 1.69	1 vs 2 vs 3: NS
Day 3 E2 (mean ± SD)	38.14 ± 15.09	31.33 ± 12.05	56.50 ± 23.33	1 vs 2 vs 3: NS
Follicular fluid volume (ml)(mean ± SD)	3.95 ± 1.95	3.63 ± 1.94	3.99 ± 2.67	1 vs 2 vs 3: NS
FSH levels in FF (IU/L)(mean ± SD)	5.021 ± 1.75	5.02 ± 2.44	5.17 ± 2.08	1 vs 2 vs 3: NS

Table 2.

ART outcomes in the three study subgroups

	Group 1: Advanced maternal age	Group 2: Implantation failure	Group 3: Implantation failure + advanced maternal age	Total
Number of ICSI attempts	40	20	18	78
Number of oocytes with PB diagnosis	156	101	56	313
Number of oocytes with an abnormal PB (%)	55 out of 156 (35.25 %)	32 out of 101 (31.68 %)	25 out of 56 (44.64 %)	112 out of 313 (35.78 %)
Number of oocytes injected with a normal PB (%)	101 out of 156 (64.75 %)	69 out of 101 (68.32 %)	31 out of 56 (45.34 %)	201 out of 313 (64.21 %)
Number of oocytes injected with no PB diagnosis	92	55	32	179
Number of two-pronucleates with a PB diagnosis	70 out of 101 (69.30 %)	47 out of 69 (68.11 %)	28 out of 31 (90.32 %)	145 out of 201 (72.14 %)
Number of embryos derived from oocytes with a PB diagnosis (%)	68 out of 70 (97.14 %)	42 out of 47 (89.36 %)	23 out of 28 (82.14 %)	133 out of 145 (91.72 %)
Number of embryos per transfer	1.97	2.59	2.06	2.14
Clinical pregnancy rate per transfer (%)	7 out of 38 (18.42 %)	4 out of 17 (23.52 %)	4 out of 16 (25.00 %)	15 out of 71 (21.12 %)

Table 3.

Patients characteristics, and stimulation characteristics for normal and abnormal PB characteristics

	FF volume (ml : mean ± SD)	Age (mean ± SD)	Day 3 basal FSH (mean ± SD)	number of oocytes retrieval (mean ± SD)	E2 serum level 36 hours before oocytes retrievel (mean ± SD)	FSH units administered per attempts (mean ± SD)
Ooytes with an abnormal PB (n = 112)	4,21 ± 2.46	37.02 ± 3.58	7.13 ± 2.75	8.01 ± 2.94	1846.188 ± 875.56	2587.05 ± 1687.93
Oocytes with a normal PB (n = 201)	3.66 ± 1.84	36.09 ± 3.83	6.75 ± 2.41	8.19 ± 3.06	2026.20 ± 781.068	2384.20 ± 1352.69
p value	*p = 0.027	*p = 0.036	p = 0.20	p = 0.61	p = 0.06	p = 0.24

Table 4.

Follicular fluid levels (LH, E2, P4, AMH, Testosterone and total protein) for normal and abnormal 1st PBs in the three subgroups

	LH (FF) mean ± SD	E2×1000 (FF) mean ± SD	P4×1000 (FF) mean ± SD	AMH (FF) mean ± SD	Testostérone (FF) mean ± SD	Total protein (FF) mean ± SD
oocytes with an abnormal PB (n = 112)	undetectable	839.50 ± 523.06	19.34 ± 8.81	1.53 ± 1.81	5.15 ± 1.94	52.41 ± 11.10
oocytes with a normal PB (n = 201)	undetectable	788.61 ± 462.64	18.12 ± 8.53	1.74 ± 2.13	5.14 ± 1.65	52.58 ± 12.34
p value		p = 0.37	p = 0.23	p = 0.38	p = 0.95	p = 0.90

Fig. 1.

Different types of FISH results in a polar body: A- Normal polar body (PB) (2 spots for each chromosome 13, 16, 18, 21, 22). B- Premature separation of sister chromatids (PSSC) with 3 spots for chromosome 16 (1 chromosome 16 and 1 super numerary chromatid 16). C- Premature separation of sister chromatids (PSSC) of chromosomes 13 and 18 with only 1 spot (only 1 chromatid) for chromosomes 13 and 18. D- Non-disjunction (ND) of chromosome 16 with 4 spots for chromosome 16 (2 chromosomes 16)

Fig. 2.

Follicular fluid FSH levels for normal and abnormal 1st PBs in the three subgroups and for the study population as a whole (Group 1: Advanced maternal age; Group 2: Implantation failure; Group 3: Implantation failure + advanced maternal age)

Fig. 3.

Number of chromosomal abnormalities in the PB (n = 313) and follicular fluid FSH levels in the three study subgroups and the study population as a whole (Group 1: Advanced maternal age; Group 2: Implantation failure; Group 3: Implantation failure + advanced maternal age)

Results

The FF properties (haemorrhagic, haematic, orange or yellow) were noted. In an initial step, we evaluated the degree of blood contamination in a series of FF samples as a function of their colour and the red blood cell count. Ten samples (3 %) were excluded because the level blood contamination was greater than 3 % (corresponding to a haemorrhagic aspect).

Patient’s characteristics and ovarian stimulation data

In Table (1) the patient’s characteristics and the ovarian stimulation data of the 3 groups are shown. Obviously, the ages are different, between group 1 (aged women) and 2 (implantation failure) (mean ± SD: 38 years and 11 months ±1 year and 11 months vs 32 years and 1 months ±2 year and 7 months; p < 0.0001) and between group 2 and 3 (mean ± SD: 32 years and 1 months ±2 year and 7 months vs 38 years and 3 months ±1 year and 7 months; p < 0.0001) but not different between group 1 and 3. The basal day 3 FSH in group 3 is higher than in group 1 and 2 (p = 0.003). The quantity of FSH administrated was lower in group 1 than in group 2 and 3 (p = 0.016). E2 levels 36 h before oocytes retrieval were also lower in group 1 and 3(implantation failure + advanced maternal age) than in group 2 but FSH levels in follicular fluid (FF) were the same in the 3 groups as well as FF volumes (5.03 ± 1.91 vs 5.25 ± 2.78 vs 5.12 ± 2.02 respectively).

ICSI and FISH results

Of the 313 oocytes diagnosed, 35.78 % (112 out of 313) were found to be abnormal in tests for chromosomes 13, 16, 18, 21 and 22. A total of 201 normal oocytes were injected and 179 were injected without a prior diagnosis (technical failure: n = 27; slightly immature, with a cytoplasmic bridge: n = 41; completely immature with no PB at the time of the biopsy and then matured at the time of the ICSI procedure: n = 111). Of the 27 technical failures, 21 PBs were lost because the cytoplasmic membrane burst during extraction and 6 were not interpretable because of a hybridization anomaly.

Groups 1, 2 and 3 did not differ significantly in terms of the first PB abnormality rates (35.25 % (55 out of 156), 31.68 % (32 out of 101) and 44.64 % (25 out of 56), respectively). The overall fertilization rate was 72.14 % (145 out of 201), with a cleavage rate of 91.72 % (133 out of 145) and a per-transfer clinical pregnancy rate of 21.12 % (15 out of 71) (Table 2).

The mechanism of the observed aneuploidies was evaluated according to the indication for PCS. Figure 1 shows different types of FISH results (normal, PCS, ND) in a polar body. In the “advanced maternal age” group (group 1), PSSC affected 74.5 % of the abnormal samples (41 out of 55) and ND affected the remaining 25.5 % (14 out of 55). In the “implantation failure” group (group 2), PSSC affected 62.5 % of the abnormal samples (20 out of 32) and ND affected 37.5 % (12 out of 32). Lastly, in group 3 (“implantation failure + advanced maternal age”), PSSC affected 56 % of the abnormal samples (14 out of 25) and ND affected 44 % (11 out of 25).

Follicular fluid and FISH results

For the study population as a whole, we found a significant difference between follicular FSH levels in normal oocytes (n = 201) and those in abnormal oocytes (n = 112) i.e. 4.85 ± 1.75 IU/L vs. 5.41 ± 2.47 IU/L, respectively; p = 0.021. In within-group analyses, this difference was only statistically significant for group 1, the group of aged woman without implantation failure and usually (80 %) with severe male infertility (4.79 ± 1.68 for 101 normal oocytes vs. 5.45 ± 1.81 IU/L for 55 abnormal oocytes; p = 0.024) (Fig. 2).

For the population as a whole, we found that a greater number of chromosomal abnormalities per oocyte (increasing from 0 to 3) was associated with a higher follicular FSH level. This trend became statistically significant for the comparison of oocytes with 0 (n = 201) and with 2 (n = 26) abnormalities per oocyte (4.85 ± 1.75 IU/L vs. 5.71 ± 1.96 IU/L, respectively; p = 0.037) in the total population. Within-group analysis primarily confirmed this elevation in group 1, where there was a significant difference in follicular FSH levels when comparing oocytes with 0 (n = 101) and 2 (n = 13) abnormalities (4.79 ± 1.68 IU/L vs. 6.01 ± 1.83 IU/L, respectively; p = 0.016). Though the number of oocytes with 3 anomalies was low (n = 7) in group 1 there was also an increased FSH level comparing oocytes with 0 (n = 101) and 3 (n = 7) abnormalities (4.97 ± 1.68 IU/L vs. 6.64 ± 2.10 IU/L, respectively; p = 0.0059) as well as comparing oocytes with 1 (n = 35) and 3 (n = 7) abnormalities (5.0 ± 1.72 IU/L vs. 6.64 ± 2.10 IU/L, respectively; p = 0.021). Due to the low number of oocytes with 3 abnormalities we grouped all the oocytes with 2 or 3 abnormalities per oocyte. This analysis presented in Fig. 2 confirmed that a greater number abnormalities per oocyte (increasing from 0 to 2 or 3) was associated with higher follicular FSH levels (Fig. 3).

Patient’s characteristics, ovarian stimulation and FISH results

As shown in Table 3 for the study as a whole we found no difference between normal and abnormal oocytes dealing with patient’s characteristics (basal FSH and indication of PCS) and dealing with stimulation characteristics (number of FSH units administrated and E2 levels 36 h before retrieval and number of oocytes). The ages of the patients were different between the normal oocytes and abnormal oocytes (p = 0.036). The follicular fluid volume was lower for the normal oocytes than the abnormal oocytes (p = 0.027)

In the within-group analysis, the elevation of FSH levels in abnormal oocytes resulting from PSSC was only found in group 1 (in which the PSSC rate was highest, at 74.5 %), with 5.57 ± 1.89 for the 41 PSSC oocytes vs. 4.79 ± 1.68 for the 101 normal oocytes, respectively (p = 0.016). In group 2, follicular FSH levels for the 12 abnormal oocytes with ND (6.93 ± 4.57 IU/L) were found to be greater than those for the 69 normal oocytes (4.76 ± 1.86 IU/L; p = 0.009) or for the 20 oocytes with PSSC (4.96 ± 2.34 IU/L; p = 0.036). In group 3, the FSH level did not appear to depend on the aetiology of the abnormality (Fig. 4).

Fig. 4.

Follicular fluid FSH levels and the mechanisms of aneuploidy (PSSC and ND) in the 3 study subgroups and the study population as a whole (Group 1: Advanced maternal age; Group 2: Implantation failure; Group 3: Implantation failure + advanced maternal age)

We did not find any other significant difference between normal and abnormal oocytes in terms of the other FF assays (E2, P4, AMH, Testosterone and total protein)(Table 4). LH was undetectable (<0.2 IU/l) in follicular fluid

Discussion

The objective of the present study was to investigate the relationship between first PB aneuploidies and the levels of various molecules in the FF (i.e. the oocyte’s microenvironment) by studying groups of women at a high risk of chromosomal abnormalities. Older patients [5, 15, 22, 25, 44–46, 69] and patients with implantation failure [7, 13, 14, 16, 70, 71] tend to have particularly high oocyte aneuploidy rates. Indeed, these two criteria are recognised as indications for pre-implantation genetic screening (PGS) for aneuploidy in many countries (but not in France, where PGD is restricted to couples in which one of the partners has a known genetic anomaly). Hence, in France, these patients undergo PCS after biopsy of the first PB. According to the French regulations, PCS must be performed on a gamete and must take place before ICSI, thus allowing only the first PB to be diagnosed.

One disadvantage of this strategy is that some oocytes cannot be screened. The main reasons are immaturity (i.e. the presence of a cytoplasmic bridge at the time of PB removal) and late maturation (between the biopsy time and ICSI time). In the present study, these factors concerned 152 oocytes (41 with a bridge and 111 without a PB at the time of the biopsy). There were also few technical failures (loss of PB and/or membrane rupture: n = 21; hybridization defects: n = 6).

These factors limit the benefit provided by this technique in terms of an increased likelihood of pregnancy. Although the safety issue must also be considered, the literature data [9, 38] and our own results are reassuring in this respect [20]. In the present study, we obtained 15 pregnancies from 71 attempts with fresh embryo transfer, i.e. a pregnancy rate of 21.12 % per transfer. This value is similar to those obtained with PGS in the same indications [37, 53, 60]. Our pregnancy rate may appear to be rather low but it must be borne in mind that our patients had a poor overall IVF prognosis.

Several randomized studies of PGS for aneuploidy screening have reported that older (over-38) patients had much the same pregnancy rate as non-screened patients [8, 22, 55, 56]. This finding may have been due to (a) technical difficulties related to mosaicism in embryos and FISH errors, (b) the potential toxicity of removal of the two blastomers (which does not seem to be the case for the PB) and/or (c) genetic normalization after diagnosis (resulting in normal blastocysts and foetuses) [6]

Polar body analysis appears to be a good strategy for overcoming these problems. Analysing the full set of chromosomes (and thus more efficient “sorting”) is possible [21, 33, 58, 73, 74] but is not technically feasible in oocytes in the few hours between follicle puncture and ICSI. Polar body array CGH was found to predict zygote ploidy with acceptable accuracy [15, 18]. A critical step seems to be the timing of the biopsy [33] and the accurate, morphological identification of the first and second polar bodies. In a recent study, the morphology of simultaneously biopsied polar bodies (though used in most studies) was not found to be predictive of the cell division origin [59]. This could explain some of the disparities in the literature concerning the origin of meiotic abnormalities. Using our five- chromosome FISH technique, the first PB abnormality rates were 35.78 % in our overall population, 35.25 % in group 1 (advanced maternal age), 31.7 % in group 2 (implantation failure) and 44.6 % in group 3 (advanced maternal age and implantation failure). These values are similar to those obtained in over-35 or over-38 patients in various series studying the first PB [65], 1998 and [30, 33, 39, 66, 69] or in patients having undergone PGS for other indications [53, 70]. Our results confirm that chromosomal abnormalities have a significant impact on implantation failure, with a frequency of 31.7 % (for the five chromosomes tested here) in a group of women with a mean age of 32 years and 1 month.

Nevertheless, the aetiology of the chromosomal abnormality appeared to differ according to the patient group - particularly if one compares patients with implantation failure with those included in the programme because of advanced maternal age only. In our series, PSSC caused 74.5 % of chromosomal abnormalities in the older patients. This finding agrees with previous reports of PSSC in 71 % of non-fertilized oocytes [43], 70 % of fertilized oocytes in women aged 35 or over [66] and 70 % of mature oocytes in women aged 38 or over [69].

In contrast, the two aetiologies had more similar frequencies in group 2 (PSSC: 62.5 %; ND: 37.5 %). These results are also similar to those reported by Pellestor [43] for under-35 patients (PSSC: 47 %; ND: 53 %) and in our previous work [70].

In the present study, we probed the relationship between oocyte aneuploidy, FSH levels in the FF and levels of AMH and the other hormones controlling ovarian function (i.e. LH, E2, P4, testosterone and inhibin B).

In order to determine hormone concentrations in the FF, we prepared dilution ranges. Total protein, FSH, LH and AMH could be assayed on pure samples, thus illustrating the need to have samples with as little blood contamination as possible. In contrast, the oestradiol and progesterone assays required prior dilution of the FF (by factors of 1:2000 and 1:1000, respectively). The sample’s appearance (ranging from yellow to haemorrhagic) prompted us to consider the problem of blood contamination and its potential impact on the hormone assays. In order to assess the degree of contamination, we evaluated the red blood cell count for each of the different aspects of FF. It turned out that contamination was almost non-existent for yellow or orange samples (with blood dilutions of 1:400 and 1:100, respectively), moderate for haematic samples and high for haemorrhagic samples. It thus makes sense to exclude haemorrhagic samples, which accounted for only 3 % of the samples.

Our results confirm the link between the age of the patient and the risk of oocyte aneuploïdy but on the other hand no predictive value of day 3 basal FSH of the patient was found (7.13 ± 2.75 vs 6.75 ± 2.41 for respectively abnormal and normal oocytes). The number of oocytes retrieved was the same for normal and abnormal oocytes. It has been suggested that elevated basal levels of endogenous FSH (associated with age and/or ovarian failure) are associated with meiotic errors [29, 42, 64]. However, Massie [35] reported that basal FSH was not predictive of fetal aneuploidy reducing the importance of endogenous FSH as a prediction of oocyte aneuploidy. Our results agree with the hypothesis that basal FSH is not predictive factor of oocyte aneuploidy. Moreover, our results suggest a link between exogenous FSH (which is required for controlled ovarian hyperstimulation before ICSI) and oocyte aneuploidy. We found significantly higher FSH levels in FF from oocytes with aneuploid PBs than in normal oocytes. We also found that the greater the number of chromosomal abnormalities per oocyte, the higher the follicular FSH level. It is clear that FSH plays a crucial role in normal follicular development. An increased volume of follicular fluid which is an indicator of the size of the follicle was also associated to an increased risk of aneuploïdy (p = 0.027) (Table 3). The hormone is used at supraphysiological high doses during ovarian hyperstimulation prior to IVF and ICSI and some authors have proposed that the high doses of gonadotropins used in ART cause oocyte aneuploidies [4, 12, 32, 40] but others have came to the opposite conclusion [10, 36, 68]. Indeed aneuploidy after ovarian controlled hyperstimulation seems to have specific characteristics. In natural conceptions aneuploidies occur mainly during the first meiosis division and in IVF over half of aneuploidies in oocytes from aged women result from second meiosis division according to [21]. The supraphysiological follicular FSH levels found in our study suggest that we were measuring exogenous hormone that was related to the quantity of FSH administered and this exogenous FSH might induce more aneuploidies than endogenous FSH.

Elevated FSH levels in FF were found associated with aneuploidy in oocyte. This could be explained if FSH either promotes the generation of abnormalities or hinder the elimination of abnormal oocytes (by blocking apoptosis, for example). Indeed, another aspect of this issue relates to the fact that FSH blocks the selection of the dominant follicle, has an anti-apoptotic effect and may thus indirectly block the putative selection of aneuploid oocytes by apoptosis leading several oocytes together to maturation. This potential effect of exogenous FSH may be visible in our group 1 (aged women), in which elevated follicular FSH may be associated with poor chromatid cohesion (i.e. PSSC, the main aetiology in group 1, at 74.5 %). This is also a group with 80 % male infertility suggesting that the women of this group have no fertility problem excepted their age. Moreover, the number of chromosomal abnormalities per PB increased with follicular FSH levels in the total population and especially so in older women - confirming the hypothesis of a harmful effect in this group.

Conversely (and somewhat surprisingly), FSH appeared to be associated with ND in women with implantation failure. In patients with implantation failure, FSH is probably a less important parameter in aneuploidy than other individual genetic or epigenetic risk factors [72]. In summary, we consider that elevated FSH may promote both oocyte abnormalities (PSSC and ND) in particularly fragile cases at risk of aneuploidy.

The other parameters tested here had little or no value as aneuploidy markers. In particular, AMH (which has been presented as a predictive marker of pregnancy [27, 54, 57]) was not significantly associated with chromosomal abnormalities.

We believe that our present results are of value in 3 main respects. First in practical terms, our results argue against excessive ovarian stimulation in ART, with the possible induction of high FSH levels in FF. In aged patients with high plasma FSH levels, it may be prejudicial to perform hyperstimulation; mild stimulation would be more appropriate. It might of course be useful to recommend PGD or prenatal diagnosis. Second measuring FSH in FF could become a non-invasive test of oocyte quality. However, the high variability in the FF from one aneuploid oocyte to another decreases this marker’s predictive value for aneuploidy. And last from a more fundamental point of view, the association of high follicular fluid FSH level and oocyte aneuploïdy suggests a role of FSH either in generating those aneuploïdies or in blocking their selection.

In terms of our understanding of the causes of chromosomal abnormalities, it may be worth focusing on the importance of hormonal changes that regulate or disrupt meiosis and thus favour aneuploidy.

Glossary

PSSC

Premature separation of sister chromatids

ND

Non-disjunction

PB

Polar body

CGH

Comparative genomic hybridization

FF

Follicular fluid

IVF

In vitro fertilization

ICSI

Intra-cytoplasmic sperm injection

PGD

Pre-implantation genetic diagnosis

FISH

Fluorescence in situ hybridization

PGS

Pre-implantation genetic screening

PCS

Preconception screening