# Polycystic ovary syndrome phenotype does not have impact on oocyte morphology.

Audrey Uk, Christine Decanter, Camille Grysole, Laura Keller, Helene Behal, Silva Mauro, Didier Dewailly, Geoffroy Robin, Anne-Laure Barbotin

► To cite this version:

Audrey Uk, Christine Decanter, Camille Grysole, Laura Keller, Helene Behal, et al. Polycystic ovary syndrome phenotype does not have impact on oocyte morphology. Reprod Biol Endocrinol, 2022, Reprod Biol Endocrinol, 20, pp.7. 10.1186/s12958-021-00874-2. hal-04387940

HAL Id: hal-04387940

https://hal.univ-lille.fr/hal-04387940

Submitted on 11 Jan 2024

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# Polycystic ovary syndrome phenotype does not have impact on oocyte morphology

Audrey Uk¹,²,<fnref n="3" />, Christine Decanter⁴,⁵, Camille Grysole⁴, Laura Keller¹, Hélène Béhal⁶, Mauro Silva², Didier Dewailly<fnref n="3" />, Geoffroy Robin<fnref n="3" />,⁴,⁵ and Anne-Laure Barbotin¹,²,<fnref n="3" />*

<figure><img src="image_2.png" /></figure>

**Abstract**

**Purpose:** The primary objective of the present study of women participating in an ICSI program was to determine whether the morphologic quality of oocytes was related to the polycystic ovary syndrome (PCOS) phenotype.

**Methods:** We performed a retrospective cohort study in the IVF unit at the Lille University Medical Center (Lille, France) between 2006 and 2015. Oocyte morphology (fragmented first polar body, abnormal zona pellucida, large perivitelline space, material in perivitelline space, abnormal shape of oocyte, granular cytoplasm and intracytoplasmic vacuoles) was evaluated in PCOS women and according to different subgroup (depending on the presence or absence of the cardinal features polycystic ovarian morphology (PCOM), hyperandrogenism (HA), and oligo-anovulation (OA)).

**Results:** A total of 1496 metaphase II oocytes (n = 602 for phenotype A combining PCOM + HA + OA, n = 462 oocytes for phenotype C: PCOM + HA, and n = 432 for phenotype D: PCOM + OA) were assessed. The phenotypes A, C and D did not differ significantly with regard to the proportion of normal oocytes (adjusted percentages (95%CI): 35.2% (31.5 to 39.1%), 25.8% (21.9 to 29.9%) and 34.0% (29.7 to 38.6%), respectively: adjusted *p* = 0.13). Likewise, there were no significant intergroup differences in oocyte morphology. The ICSI outcome was not significantly associated with the PCOS phenotype.

**Conclusion:** The present study is the first to show that the PCOS phenotype (notably the presence vs. absence of OA and/or HA) is not significantly associated with the morphological quality of oocytes.

**Keywords:** Polycystic ovary syndrome, PCOS phenotypes, Oocyte morphology, Morphologic scores, ICSI

**Introduction**

Polycystic ovary syndrome (PCOS) is the most common endocrine reproductive disorder; worldwide, it affects 5-20% of women of reproductive age [1]. According to the Rotterdam consensus criteria and after the exclusion of related disorders, the diagnosis of PCOS is based on the presence of at least two the following cardinal features: oligo-ovulation/anovulation (OA), clinical and/or biochemical hyperandrogenism (HA), and a polycystic

ovarian morphology (PCOM) on ultrasound with >20 follicles per ovary [2–4]. Depending on the presence or absence of these cardinal features, a case of PCOS is then classified as phenotype A, B, C or D [2, 5]. PCOS phenotype A is characterized by the presence of all three features (i.e. OA + HA + PCOM), whereas the other phenotypes are characterized by the presence of only two of the features.

In vitro fertilization (IVF) may be required for women with anovulatory PCOS and who have failed to become pregnant with ovulation induction or when there are additional infertility factors, such as tubal stenosis or male subfertility [4, 6, 7]. It has been postulated that PCOS women produce more oocytes than healthy

<footnote>*Correspondence: anne-laure.barbotin@chru-lille.fr</footnote>

<footnote n="3">³ Department of Medicine, University of Lille, Lille, France
Full list of author information is available at the end of the article</footnote>
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# Polycystic ovary syndrome phenotype does not have impact on oocyte morphology

Audrey Uk¹,²,<fnref n="3" />, Christine Decanter⁴,⁵, Camille Grysole⁴, Laura Keller¹, Hélène Béhal⁶, Mauro Silva², Didier Dewailly<fnref n="3" />, Geoffroy Robin<fnref n="3" />,⁴,⁵ and Anne-Laure Barbotin¹,²,<fnref n="3" />*

<figure><img src="image_3.png" /></figure>

**Abstract**

**Purpose:** The primary objective of the present study of women participating in an ICSI program was to determine whether the morphologic quality of oocytes was related to the polycystic ovary syndrome (PCOS) phenotype.

**Methods:** We performed a retrospective cohort study in the IVF unit at the Lille University Medical Center (Lille, France) between 2006 and 2015. Oocyte morphology (fragmented first polar body, abnormal zona pellucida, large perivitelline space, material in perivitelline space, abnormal shape of oocyte, granular cytoplasm and intracytoplasmic vacuoles) was evaluated in PCOS women and according to different subgroup (depending on the presence or absence of the cardinal features polycystic ovarian morphology (PCOM), hyperandrogenism (HA), and oligo-anovulation (OA)).

**Results:** A total of 1496 metaphase II oocytes (n = 602 for phenotype A combining PCOM + HA + OA, n = 462 oocytes for phenotype C: PCOM + HA, and n = 432 for phenotype D: PCOM + OA) were assessed. The phenotypes A, C and D did not differ significantly with regard to the proportion of normal oocytes (adjusted percentages (95%CI): 35.2% (31.5 to 39.1%), 25.8% (21.9 to 29.9%) and 34.0% (29.7 to 38.6%), respectively: adjusted *p* = 0.13). Likewise, there were no significant intergroup differences in oocyte morphology. The ICSI outcome was not significantly associated with the PCOS phenotype.

**Conclusion:** The present study is the first to show that the PCOS phenotype (notably the presence vs. absence of OA and/or HA) is not significantly associated with the morphological quality of oocytes.

**Keywords:** Polycystic ovary syndrome, PCOS phenotypes, Oocyte morphology, Morphologic scores, ICSI

**Introduction**

Polycystic ovary syndrome (PCOS) is the most common endocrine reproductive disorder; worldwide, it affects 5-20% of women of reproductive age [1]. According to the Rotterdam consensus criteria and after the exclusion of related disorders, the diagnosis of PCOS is based on the presence of at least two the following cardinal features: oligo-ovulation/anovulation (OA), clinical and/or biochemical hyperandrogenism (HA), and a polycystic

ovarian morphology (PCOM) on ultrasound with >20 follicles per ovary [2–4]. Depending on the presence or absence of these cardinal features, a case of PCOS is then classified as phenotype A, B, C or D [2, 5]. PCOS phenotype A is characterized by the presence of all three features (i.e. OA + HA + PCOM), whereas the other phenotypes are characterized by the presence of only two of the features.

In vitro fertilization (IVF) may be required for women with anovulatory PCOS and who have failed to become pregnant with ovulation induction or when there are additional infertility factors, such as tubal stenosis or male subfertility [4, 6, 7]. It has been postulated that PCOS women produce more oocytes than healthy

<footnote>*Correspondence: anne-laure.barbotin@chru-lille.fr</footnote>

<footnote n="3">³ Department of Medicine, University of Lille, Lille, France
Full list of author information is available at the end of the article</footnote>
women in response to controlled ovarian hyperstimu-
lation (COH) [8–11]. However, it is also suspected that
these PCOS oocytes may be of poor quality as a result of
intra- and extra-ovarian factors [12]; this might lead to
a lower fertilization rate, poor embryo quality, a lower
implantation rate and a higher miscarriage rate [8–10,
13, 14]. Indeed, it is well known that oocyte competence
influences embryonic development [15]. To date, only a
few studies have assessed the potential impact of different
PCOS phenotypes on the outcomes of assisted reproduc-
tive technologies [16–19]. In a study of IVF procedures,
Ramezanali and colleagues (2016) reported lower clini-
cal pregnancy rates in PCOS phenotypes A and B than in
controls - suggesting that the combination of HA and OA
may affect embryonic development [18]. Furthermore,
the risk of miscarriage was greater for phenotypes A, B
and D than for phenotype C (ovulatory PCOS) [16]. The
heterogeneity of the PCOS study populations in these
reports (i.e. interstudy differences in the inclusion crite-
ria and a mixture of IVF and ICSI procedures) makes it
difficult to draw definitive conclusions. Nevertheless, it is
likely that the oocytes are of poor morphologic quality in
cases of full-blown PCOS. Furthermore, several studies
have found that the oocyte's developmental competence
might be impaired in women with PCOS [12, 20]. In par-
ticular, it has been suggested that the oocyte's morpho-
logic quality is linked to its implantation potential [21,
22]. On the one hand, Alikani and colleagues reported
reduced pregnancy and implantation rates in women
with exclusive replacement of embryos, originating from
dysmorphic oocytes regardless of the oocyte abnormal-
ity features [21]. On the other hand, Kahraman and col-
leagues have suggested that the oocytes with severe
central granulation in their cytoplasm seem to have less
implantation and on-going pregnancy potential [22].
Oocyte quality is a key limiting factor in female fertility;
it reflects the oocyte's intrinsic developmental potential
and has an essential role in fertilization and subsequent
development [23].

An oocyte's quality is primarily reflected by its mor-
phologic features. For instance, centrally located cyto-
plasm granulations are associated with low fertilization
rates [24], poor pregnancy outcomes, and low live birth
and miscarriage rates in ICSI programs [25]. However, lit-
tle is known about the relationship between oocyte mor-
phology and the PCOS phenotype. Indeed, the influence
of the oocyte's competence on the reproductive potential
of women with PCOS depends on the phenotype and the
related comorbidities [15]. Thus, the primary objective
of the present study of women in an ICSI program was
to determine whether oocyte morphologic quality var-
ies from one PCOS phenotype to another. To this end,
we used the criteria routinely applied in IVF centers to

evaluate the oocytes' morphological features. The study's
secondary objective was to determine whether the oocyte
morphology differs when comparing women with PCOS,
women with PCOM only, and other (control) women in
an ICSI program.

**Material and methods**

Study design and population

Data on clinical, endocrine and ultrasound features
were extracted from a computerized database compiled
between January 2006 and December 2015 in the IVF
center at Lille University Medical Center (Lille, France).
Women having participated in an ICSI program were
included retrospectively. The study results were reported
in compliance with the STROBE statement [26]. In line
with the French legislation on retrospective studies of
routine clinical practice, the study protocol was approved
by the Lille hospital committee.

For the PCOS group, the inclusion criteria were (i) age 18–38, (ii) a confirmed diagnosis of PCOS (see below); (iii) the presence of at least one COH procedure followed by successful oocyte retrieval; and (iv) inclusion in an ICSI program in a sole indication of male infertility. The non-inclusion criteria were (i) previous pelvic or ovarian surgery, (ii) previous or ongoing endocrine disorders (other than PCOS), and (iii) use of medications known to have an influence on the endocrine profile. The following exclusion criteria were: age<18 or more than 38 years, a low suspected ovarian reserve [27] (FSH>12 IU/l), pregnant or breastfeeding women, and hormonal contraceptive users. We excluded women with differential diagnosis for PCOS: hyperprolactinemia (serum prolactin >20 ng/ml on two separate determinations), functionnal hypothalamic amenorrhea, other congenital or acquired gonadotropic deficiencies, nonclassic 21-hydroxylase deficiency (basal 17-hydroxyprogesterone (17-OHP) >5 ng/ml and/or post-adrenocorticotrophic hormone-stimulated value >12 ng/ml). Ovarian or adrenal tumours were excluded on the basis of serum total testosterone (TT) or dehydroepiandrosterone sulfate levels lower than 1.5 ng/ml or 15 µmol/l, respectively. Moreover, any patient with at least one follicle with a diameter>9 mm at U/S or a serum estradiol (E2) level above 80 pg/ml was excluded from the study. Women with a history of pelvic or ovarian surgery, severe endometriosis, anovulation and/or hyperandrogenism not related to PCOS were also excluded from the study.

All patients were diagnosed with PCOS (according to
the modified Rotterdam classification) and so fulfilled
at least two of the following three criteria: OA, clini-
cal and/or biochemical HA, and PCOM (defined as ≥19
follicles per ovary) and/or an excessive serum anti-Mül-
lerian hormone (AMH) level ≥ 35 pmol/l), as previously
women in response to controlled ovarian hyperstimulation (COH) [8–11]. However, it is also suspected that these PCOS oocytes may be of poor quality as a result of intra- and extra-ovarian factors [12]; this might lead to a lower fertilization rate, poor embryo quality, a lower implantation rate and a higher miscarriage rate [8–10, 13, 14]. Indeed, it is well known that oocyte competence influences embryonic development [15]. To date, only a few studies have assessed the potential impact of different PCOS phenotypes on the outcomes of assisted reproductive technologies [16–19]. In a study of IVF procedures, Ramezanali and colleagues (2016) reported lower clinical pregnancy rates in PCOS phenotypes A and B than in controls - suggesting that the combination of HA and OA may affect embryonic development [18]. Furthermore, the risk of miscarriage was greater for phenotypes A, B and D than for phenotype C (ovulatory PCOS) [16]. The heterogeneity of the PCOS study populations in these reports (i.e. interstudy differences in the inclusion criteria and a mixture of IVF and ICSI procedures) makes it difficult to draw definitive conclusions. Nevertheless, it is likely that the oocytes are of poor morphologic quality in cases of full-blown PCOS. Furthermore, several studies have found that the oocyte's developmental competence might be impaired in women with PCOS [12, 20]. In particular, it has been suggested that the oocyte's morphologic quality is linked to its implantation potential [21, 22]. On the one hand, Alikani and colleagues reported reduced pregnancy and implantation rates in women with exclusive replacement of embryos, originating from dysmorphic oocytes regardless of the oocyte abnormality features [21]. On the other hand, Kahraman and colleagues have suggested that the oocytes with severe central granulation in their cytoplasm seem to have less implantation and on-going pregnancy potential [22]. Oocyte quality is a key limiting factor in female fertility; it reflects the oocyte's intrinsic developmental potential and has an essential role in fertilization and subsequent development [23].

An oocyte's quality is primarily reflected by its morphologic features. For instance, centrally located cytoplasm granulations are associated with low fertilization rates [24], poor pregnancy outcomes, and low live birth and miscarriage rates in ICSI programs [25]. However, little is known about the relationship between oocyte morphology and the PCOS phenotype. Indeed, the influence of the oocyte's competence on the reproductive potential of women with PCOS depends on the phenotype and the related comorbidities [15]. Thus, the primary objective of the present study of women in an ICSI program was to determine whether oocyte morphologic quality varies from one PCOS phenotype to another. To this end, we used the criteria routinely applied in IVF centers to

evaluate the oocytes' morphological features. The study's secondary objective was to determine whether the oocyte morphology differs when comparing women with PCOS, women with PCOM only, and other (control) women in an ICSI program.

# Material and methods

## Study design and population

Data on clinical, endocrine and ultrasound features were extracted from a computerized database compiled between January 2006 and December 2015 in the IVF center at Lille University Medical Center (Lille, France). Women having participated in an ICSI program were included retrospectively. The study results were reported in compliance with the STROBE statement [26]. In line with the French legislation on retrospective studies of routine clinical practice, the study protocol was approved by the Lille hospital committee.

For the PCOS group, the inclusion criteria were (i) age 18–38, (ii) a confirmed diagnosis of PCOS (see below); (iii) the presence of at least one COH procedure followed by successful oocyte retrieval; and (iv) inclusion in an ICSI program in a sole indication of male infertility. The non-inclusion criteria were (i) previous pelvic or ovarian surgery, (ii) previous or ongoing endocrine disorders (other than PCOS), and (iii) use of medications known to have an influence on the endocrine profile. The following exclusion criteria were: age < 18 or more than 38 years, a low suspected ovarian reserve [27] (FSH > 12 IU/l), pregnant or breastfeeding women, and hormonal contraceptive users. We excluded women with differential diagnosis for PCOS: hyperprolactinemia (serum prolactin > 20 ng/ml on two separate determinations), functional hypothalamic amenorrhea, other congenital or acquired gonadotropic deficiencies, nonclassic 21-hydroxylase deficiency (basal 17-hydroxyprogesterone (17-OHP) > 5 ng/ml and/or post-adrenocorticotrophic hormone-stimulated value > 12 ng/ml). Ovarian or adrenal tumours were excluded on the basis of serum total testosterone (TT) or dehydroepiandrosterone sulfate levels lower than 1.5 ng/ml or 15 µmol/l, respectively. Moreover, any patient with at least one follicle with a diameter > 9 mm at U/S or a serum estradiol (E2) level above 80 pg/ml was excluded from the study. Women with a history of pelvic or ovarian surgery, severe endometriosis, anovulation and/or hyperandrogenism not related to PCOS were also excluded from the study.

All patients were diagnosed with PCOS (according to the modified Rotterdam classification) and so fulfilled at least two of the following three criteria: OA, clinical and/or biochemical HA, and PCOM (defined as ≥ 19 follicles per ovary) and/or an excessive serum anti-Müllerian hormone (AMH) level ≥ 35 pmol/l), as previously
reported [28, 29]. Clinical HA was defined as the presence of hirsutism (with a modified Ferriman–Gallwey score ≥ 6) and/or acne in more than two body areas [30]. Biochemical HA was defined as serum total testosterone (T) and/or androstenedione level above the 95th percentile value for controls with normal cycles (> 0.53 ng/mL and > 2.07 ng/mL, respectively) [29]. OA was defined as amenorrhea or a cycle length above 35 days.

We included a total of 65 women in the PCOM-only group. Indeed, some researchers consider that PCOM is part of a complex PCOS spectrum disorder, with abnormal granulosa cell activity [31, 32]. The inclusion criteria for this group were (i) age 18–38, (ii) PCOM defined as either an ovarian volume ≥ 10 ml, an ovarian surface area ≥ 5.5 cm², a follicle number per ovary (FNPO) ≥ 19 or an AMH level ≥ 35 pmol/l or both [28], (iii) the presence of at least one COH procedure followed by successful oocyte retrieval; and (iv) inclusion in an ICSI program in a sole indication of male infertility. The following exclusion criteria were: age < 18 or more than 38 years, a low suspected ovarian reserve [27], pregnant or breastfeeding women, any additional Rotterdam criteria (i.e. HA and/or OA), hormonal contraceptive users, previous pelvic or ovarian surgery and any risk factors for infertility (pelvic surgery, endometriosis etc) and use of medications known to have an influence on the endocrine profile.

For the control group comprising 58 women, the inclusion criteria were (i) age 18–38, (ii) ovulatory cycles, no signs of hyperandrogenism, an FNPO between 8 and 18 follicles in each ovary, FSH < 10 IU/L, and AMH < 35 pmol/L, (iii) the presence of at least one COH procedure followed by successful oocyte retrieval; and (iv) inclusion in an ICSI program in a sole indication of male infertility. The following exclusion criteria were: age < 18 or more than 38 years, a low suspected ovarian reserve [27], pregnant or breastfeeding women, and hormonal contraceptive users, (previous pelvic or ovarian surgery, any Rotterdam criteria or risk factors for infertility (pelvic surgery, endometriosis etc.) and use of medications known to have an influence on the endocrine profile.

The 110 women diagnosed with PCOS were classified into four phenotype subgroups (Table 1). As there was only one woman with a PCOS B phenotype, we excluded her (for reasons of representativeness) from the study population. Hence, 109 women were included in the final analysis (PCOS A: 41 patients and 77 cycles; PCOS C: 31 patients and 57 cycles; PCOS D: 37 patients and 55 cycles). A total of 189 ICSI treatment cycles were analyzed during the study period. In all cases, male factor infertility indicated ICSI with fresh ejaculated spermatotozoa only. ICSI cycles using surgically acquired sperm were excluded. Additionally, only ICSI cycles following sperm processing using density gradient for sperm selection were included in the study.

## Laboratory tests and ultrasound examinations

All patients had a baseline endocrine screen and ultrasound examination on the same day during the early follicular phase (i.e. between days 2 and 5). In patients with OA, the menstrual period was either spontaneous or induced by the administration of dydrogesterone. Serum FSH and estradiol (E2) levels were measured using chemiluminescent, two-site immunoassays on a multiparameter system (Axsym®; Abbott Laboratories, Chicago, IL, USA). Serum AMH levels were measured using a second-generation enzyme immunoassay (A16507, Beckman Coulter, Immunotech, Villepinte, France). Delta-4-androstenedione and total testosterone levels were measured in duplicate using a radioimmunoassay, as described previously [33]. The baseline FNPO assessment was performed with a Voluson E8 Expert system (General Electric Systems®) and a 5–9 MHz transvaginal transducer, by counting all the follicles with a diameter of 2 to 9 mm, as described previously [28, 34]. The mean follicle count for the left and right ovaries was used in the statistical analysis.

## The COH protocol

All patients underwent a gonadotropin-releasing hormone (GnRH) agonist or antagonist protocol. In the agonist protocol, daily injections with triptorelin (0.1 mg) were initiated 1 week before the expected start of the

<table><caption>**Table 1** Criteria defining the four PCOS phenotypes</caption><thead><tr><th rowspan="2">Modified Rotterdam Criteria</th><th colspan="4">Polycystic ovary syndrome</th></tr><tr><th>Phenotype A</th><th>Phenotype B</th><th>Phenotype C</th><th>Phenotype D</th></tr></thead><tbody><tr><td>Oligoanovulation</td><td>Yes</td><td>Yes</td><td>No</td><td>Yes</td></tr><tr><td>Clinical and/or biochemical hyperandrogenism</td><td>Yes</td><td>Yes</td><td>Yes</td><td>No</td></tr><tr><td>PCOM, AMH ≥35 pmol/l, or both</td><td>Yes</td><td>No</td><td>Yes</td><td>Yes</td></tr></tbody></table>
reported [28, 29]. Clinical HA was defined as the presence of hirsutism (with a modified Ferriman–Gallwey score ≥ 6) and/or acne in more than two body areas [30]. Biochemical HA was defined as serum total testosterone (T) and/or androstenedione level above the 95th percentile value for controls with normal cycles (> 0.53 ng/mL and > 2.07 ng/mL, respectively) [29]. OA was defined as amenorrhea or a cycle length above 35 days.

We included a total of 65 women in the PCOM-only group. Indeed, some researchers consider that PCOM is part of a complex PCOS spectrum disorder, with abnormal granulosa cell activity [31, 32]. The inclusion criteria for this group were (i) age 18–38, (ii) PCOM defined as either an ovarian volume ≥ 10 ml, an ovarian surface area ≥ 5.5 cm², a follicle number per ovary (FNPO) ≥ 19 or an AMH level ≥ 35 pmol/l or both [28], (iii) the presence of at least one COH procedure followed by successful oocyte retrieval; and (iv) inclusion in an ICSI program in a sole indication of male infertility. The following exclusion criteria were: age < 18 or more than 38 years, a low suspected ovarian reserve [27], pregnant or breastfeeding women, any additional Rotterdam criteria (i.e. HA and/or OA), hormonal contraceptive users, previous pelvic or ovarian surgery and any risk factors for infertility (pelvic surgery, endometriosis etc) and use of medications known to have an influence on the endocrine profile.

For the control group comprising 58 women, the inclusion criteria were (i) age 18–38, (ii) ovulatory cycles, no signs of hyperandrogenism, an FNPO between 8 and 18 follicles in each ovary, FSH < 10 IU/L, and AMH < 35 pmol/L, (iii) the presence of at least one COH procedure followed by successful oocyte retrieval; and (iv) inclusion in an ICSI program in a sole indication of male infertility. The following exclusion criteria were: age < 18 or more than 38 years, a low suspected ovarian reserve [27], pregnant or breastfeeding women, and hormonal contraceptive users, (previous pelvic or ovarian surgery, any Rotterdam criteria or risk factors for infertility (pelvic surgery, endometriosis etc.) and use of medications known to have an influence on the endocrine profile.

The 110 women diagnosed with PCOS were classified into four phenotype subgroups (Table 1). As there was only one woman with a PCOS B phenotype, we excluded her (for reasons of representativeness) from the study population. Hence, 109 women were included in the final analysis (PCOS A: 41 patients and 77 cycles; PCOS C: 31 patients and 57 cycles; PCOS D: 37 patients and 55 cycles). A total of 189 ICSI treatment cycles were analyzed during the study period. In all cases, male factor infertility indicated ICSI with fresh ejaculated spermatotozoa only. ICSI cycles using surgically acquired sperm were excluded. Additionally, only ICSI cycles following sperm processing using density gradient for sperm selection were included in the study.

## Laboratory tests and ultrasound examinations

All patients had a baseline endocrine screen and ultrasound examination on the same day during the early follicular phase (i.e. between days 2 and 5). In patients with OA, the menstrual period was either spontaneous or induced by the administration of dydrogesterone. Serum FSH and estradiol (E2) levels were measured using chemiluminescent, two-site immunoassays on a multiparameter system (Axsym®; Abbott Laboratories, Chicago, IL, USA). Serum AMH levels were measured using a second-generation enzyme immunoassay (A16507, Beckman Coulter, Immunotech, Villepinte, France). Delta-4-androstenedione and total testosterone levels were measured in duplicate using a radioimmunoassay, as described previously [33]. The baseline FNPO assessment was performed with a Voluson E8 Expert system (General Electric Systems®) and a 5–9 MHz transvaginal transducer, by counting all the follicles with a diameter of 2 to 9 mm, as described previously [28, 34]. The mean follicle count for the left and right ovaries was used in the statistical analysis.

## The COH protocol

All patients underwent a gonadotropin-releasing hormone (GnRH) agonist or antagonist protocol. In the agonist protocol, daily injections with triptorelin (0.1 mg) were initiated 1 week before the expected start of the

<table><caption>**Table 1** Criteria defining the four PCOS phenotypes</caption><thead><tr><th rowspan="2">Modified Rotterdam Criteria</th><th colspan="4">Polycystic ovary syndrome</th></tr><tr><th>Phenotype A</th><th>Phenotype B</th><th>Phenotype C</th><th>Phenotype D</th></tr></thead><tbody><tr><td>Oligoanovulation</td><td>Yes</td><td>Yes</td><td>No</td><td>Yes</td></tr><tr><td>Clinical and/or biochemical hyperandrogenism</td><td>Yes</td><td>Yes</td><td>Yes</td><td>No</td></tr><tr><td>PCOM, AMH ≥35 pmol/l, or both</td><td>Yes</td><td>No</td><td>Yes</td><td>Yes</td></tr></tbody></table>
following cycle. Desensitization was checked 12-15 days after initiation of the agonist protocol. When the E2 concentration was below 50 pg/mL and an ultrasound examination had confirmed the absence of functional ovarian cysts, COH was initiated with daily injections of recombinant FSH (rFSH) or human menopausal gonadotropin (HMG). In the antagonist protocol, women received daily injections of gonadotropins (rFSH or HMG) starting on day 2 of menstrual bleeding, followed by treatment with a GnRH antagonist (Ganirelix/Orgalutran®, MSD®, Courbevoie, France) starting on day 6 of the rFSH treatment. The starting dose of FSH ranged from 75 to 225 IU/d, depending on the body mass index (BMI), the antral follicle count, and the serum AMH level. The FSH dose was then set individually as a function of the E2 level and follicular growth during COH. Recombinant human chorionic gonadotropin (hCG, Ovitrelle®, Merck, Lyon, France) was administered when at least three follicles with a diameter > 17 mm were observed on ultrasound at the same time as a consistent rise in the serum E2 level. Oocytes were retrieved (using transvaginal ultrasound-guided needle aspiration) 36 h after the hCG injection.

## ICSI procedure and assessment of oocyte morphology

On the day of the ICSI, a semen sample was obtained by masturbation and collected in a sterile container. Sperm processing was performed with two discontinuous layers of PureSperm® (JCD, La Mulatière, France). After semen liquefaction, 1 to 2 mL of the ejaculates were layered in a sterile Falcon tube and centrifuged for 20 min at 300 x g. Following centrifugation, the supernatant was aspirated and discarded. Next, pellets containing selected spermatozoa were aspirated and transferred into new sterile Falcon tubes containing washing medium (Ferticult Hepes®, JCD, La Mulatière, France) and centrifuged at 300×g for 10 min. Pellet were re-suspended in 100 µL of Ferticult Hepes® medium and used for ICSI procedure. Sperm concentration and progressive sperm motility in the final preparation were assessed in the final preparation.

Two hours after the oocyte retrieval, the oocytes were stripped using chemical and mechanical methods. Each cumulus-oocyte complex was plunged for 20 s into a hyaluronidase solution (80 IU/mL, FertiPro®, France), following by successive aspirations and expulsions through a micropipette to remove the cumulus and the corona radiata cells. The oocytes' meiotic maturity and morphology were evaluated by two independent operators at the time of the ICSI, using an inverted microscope (Leica DMIRB, Leica Microsystems®, Germany) and a magnification of × 400. A metaphase II oocyte was considered

as morphologically normal when it exhibited a clear, homogenous cytoplasm with a uniform texture, with a smooth surface (with no vacuoles, smooth endoplasmic reticulum or granulations), a round-clear zona pellucida, with perivitelline space of normal size containing a single-non fragmented, round or ovoid, normal-sized first polar body.

The oocytes were screened for extracytoplasmic and intracytoplasmic abnormalities. The possible extracytoplasmic morphologic abnormalities were as follows: a fragmented or abnormal first polar body, an abnormal zona pellucida (thick, fine, or irregular), the presence of a large perivitelline space (PVS), the presence of material in the PVS, and an abnormally shaped oocyte. The possible intracytoplasmic morphological abnormalities included an abnormally granular cytoplasm (heterogeneous granulations or granulations concentrated in a central zone) and the presence of one or more vacuoles. In accordance with international guidelines, huge oocytes and/or oocytes containing inclusions of smooth endoplasmic reticulum were not injected [35]. Two oocyte morphology scores were determined: the average oocyte quality index (AOQI) [36] and the metaphase (M)II oocyte morphologic score (MOMS) [37]. The AOQI is calculated by first rating each oocyte for the presence (score: 1) or absence (score: 0) of each of the following morphologic abnormalities: granular cytoplasm, an abnormal zona pellucida, a fragmented or abnormal polar body, a large PVS, material in the PVS, an abnormal shape, and vacuoles. The AOQI then corresponds to the ratio between the total number of abnormalities counted and the total number of MII oocytes. The MOMS quantifies the severity of the morphologic characteristics in relation to the associated ICSI outcome; oocytes with a low MOMS rate have a greater implantation potential than those with a high MOMS rate (Table 2).

As reported previously, our evaluation of morphologic oocyte quality is highly reproducible (as judged by the level of inter-operator agreement) [36].

<table><caption>**Table 2** Metaphase II oocyte morphological score (MOMS, from Rienzi et al., 2008 [37])</caption><thead><tr><th></th><th>Points</th></tr></thead><tbody><tr><td><strong>Extracytoplasmic features</strong></td><td></td></tr><tr><td>Abnormal first polar body</td><td>2.0</td></tr><tr><td>Large perivitelline space</td><td>1.4</td></tr><tr><td><strong>Cytoplasmic features</strong></td><td></td></tr><tr><td>Granular cytoplasm</td><td>1.4</td></tr><tr><td>Centrally located granular area</td><td>2.7</td></tr><tr><td>Vacuoles</td><td>2.1</td></tr></tbody></table>
following cycle. Desensitization was checked 12-15 days after initiation of the agonist protocol. When the E2 concentration was below 50 pg/mL and an ultrasound examination had confirmed the absence of functional ovarian cysts, COH was initiated with daily injections of recombinant FSH (rFSH) or human menopausal gonadotropin (HMG). In the antagonist protocol, women received daily injections of gonadotropins (rFSH or HMG) starting on day 2 of menstrual bleeding, followed by treatment with a GnRH antagonist (Ganirelix/Orgalutran®, MSD®, Courbevoie, France) starting on day 6 of the rFSH treatment. The starting dose of FSH ranged from 75 to 225 IU/d, depending on the body mass index (BMI), the antral follicle count, and the serum AMH level. The FSH dose was then set individually as a function of the E2 level and follicular growth during COH. Recombinant human chorionic gonadotropin (hCG, Ovitrelle®, Merck, Lyon, France) was administered when at least three follicles with a diameter > 17 mm were observed on ultrasound at the same time as a consistent rise in the serum E2 level. Oocytes were retrieved (using transvaginal ultrasound-guided needle aspiration) 36 h after the hCG injection.

## ICSI procedure and assessment of oocyte morphology

On the day of the ICSI, a semen sample was obtained by masturbation and collected in a sterile container. Sperm processing was performed with two discontinuous layers of PureSperm® (JCD, La Mulatière, France). After semen liquefaction, 1 to 2 mL of the ejaculates were layered in a sterile Falcon tube and centrifuged for 20 min at 300 x g. Following centrifugation, the supernatant was aspirated and discarded. Next, pellets containing selected spermatozoa were aspirated and transferred into new sterile Falcon tubes containing washing medium (Ferticult Hepes®, JCD, La Mulatière, France) and centrifuged at 300×g for 10 min. Pellet were re-suspended in 100 µL of Ferticult Hepes® medium and used for ICSI procedure. Sperm concentration and progressive sperm motility in the final preparation were assessed in the final preparation.

Two hours after the oocyte retrieval, the oocytes were stripped using chemical and mechanical methods. Each cumulus-oocyte complex was plunged for 20 s into a hyaluronidase solution (80 IU/mL, FertiPro®, France), following by successive aspirations and expulsions through a micropipette to remove the cumulus and the corona radiata cells. The oocytes' meiotic maturity and morphology were evaluated by two independent operators at the time of the ICSI, using an inverted microscope (Leica DMIRB, Leica Microsystems®, Germany) and a magnification of × 400. A metaphase II oocyte was considered

as morphologically normal when it exhibited a clear, homogenous cytoplasm with a uniform texture, with a smooth surface (with no vacuoles, smooth endoplasmic reticulum or granulations), a round-clear zona pellucida, with perivitelline space of normal size containing a single-non fragmented, round or ovoid, normal-sized first polar body.

The oocytes were screened for extracytoplasmic and intracytoplasmic abnormalities. The possible extracytoplasmic morphologic abnormalities were as follows: a fragmented or abnormal first polar body, an abnormal zona pellucida (thick, fine, or irregular), the presence of a large perivitelline space (PVS), the presence of material in the PVS, and an abnormally shaped oocyte. The possible intracytoplasmic morphological abnormalities included an abnormally granular cytoplasm (heterogeneous granulations or granulations concentrated in a central zone) and the presence of one or more vacuoles. In accordance with international guidelines, huge oocytes and/or oocytes containing inclusions of smooth endoplasmic reticulum were not injected [35]. Two oocyte morphology scores were determined: the average oocyte quality index (AOQI) [36] and the metaphase (M)II oocyte morphologic score (MOMS) [37]. The AOQI is calculated by first rating each oocyte for the presence (score: 1) or absence (score: 0) of each of the following morphologic abnormalities: granular cytoplasm, an abnormal zona pellucida, a fragmented or abnormal polar body, a large PVS, material in the PVS, an abnormal shape, and vacuoles. The AOQI then corresponds to the ratio between the total number of abnormalities counted and the total number of MII oocytes. The MOMS quantifies the severity of the morphologic characteristics in relation to the associated ICSI outcome; oocytes with a low MOMS rate have a greater implantation potential than those with a high MOMS rate (Table 2).

As reported previously, our evaluation of morphologic oocyte quality is highly reproducible (as judged by the level of inter-operator agreement) [36].

<table><caption>**Table 2** Metaphase II oocyte morphological score (MOMS, from Rienzi et al., 2008 [37])</caption><thead><tr><th></th><th>Points</th></tr></thead><tbody><tr><td><strong>Extracytoplasmic features</strong></td><td></td></tr><tr><td>Abnormal first polar body</td><td>2.0</td></tr><tr><td>Large perivitelline space</td><td>1.4</td></tr><tr><td><strong>Cytoplasmic features</strong></td><td></td></tr><tr><td>Granular cytoplasm</td><td>1.4</td></tr><tr><td>Centrally located granular area</td><td>2.7</td></tr><tr><td>Vacuoles</td><td>2.1</td></tr></tbody></table>
## Evaluation of embryos and ICSI cycles

Each MII oocyte was microinjected as part of a ICSI procedure described previously [38]. Fertilization (the presence of two distinct pronuclei) was scored 16-18 h after injection. Morphological embryo quality (i.e. the number of blastomeres, cell symmetry and fragmentation) was evaluated on day 2 or 3 according to the European Society of Human Reproduction and Embryology classification [35] A “top-quality embryo” was defined as the presence of four blastomeres on day 2 or eight blastomeres on day 3, symmetrical cleavage, and a fragmentation rate below 10% [35]. Top-quality embryos were scored as grade 1, and poor-quality embryos were scored as grade 3. Supernumerary top-quality embryos were frozen for subsequent transfer. Ultrasound-guided embryo transfer was performed 2 or 3 days after oocyte retrieval.

Luteal phase support (vaginal micronized progesterone 200 mg, 3 times a day) was indicated for all patients and was initiated on the evening of oocyte retrieval. The small proportion of freeze-all cycles (no fresh embryo transfers) was not taken into account in the comparisons of the ICSI outcomes.

## Outcomes

For the PCOS, PCOM-only and control groups, oocyte quality and morphology were assessed at the time of ICSI injection with regard to the oocyte maturity index (defined as the ratio between the number of MII oocytes and the total number of oocytes collected), the seven extracytoplasmic and intracytoplasmic abnormalities mentioned above, the AOQI, and the MOMS were performed.

After excluding cycles using frozen-thawed sperm, we evaluated the fertilization rate (FR), implantation rate (IR), clinical pregnancy rate (CPR), live birth rate (LBR) per transfer for day 2-3 embryos in fresh cycles, miscarriage rate (MR), and cumulative CPR. The IR was defined as the ratio between the number of gestational sacs with fetal heart activity and the number of embryos transferred. The live birth rate was defined as the number of live births per embryo transfer in fresh cycles. The cumulative CPR pregnancy rate was defined as the number of pregnancies with at least one gestational sac exhibiting fetal heart activity, including embryo transfers in fresh cycles and frozen-thawed cycles performed between 2006 and 2016.

## Statistical analysis

Quantitative variables were expressed as the mean ± standard deviation (for normally distributed datasets) or as the median [interquartile range (IQR)] otherwise. Normality of distributions was checked graphically and using Shapiro Wilk test. Categorical

variables were expressed by frequencies and percentages. Comparisons of subject's characteristics (at the first cycle) between the three PCOS phenotypes were performed with Chi-square test for current smoker women; and with analysis of variance for normally distributed quantitative variables or Kruskal-Wallis test otherwise. Post-hoc pairwise comparisons were performed in case of global significant difference and a Bonferroni correction was applied.

Groups were compared (i.e. PCOS vs PCOM-only vs controls; PCOS phenotypes A vs. C vs. D) using generalized estimating equation (GEE) models (to account for the correlation between repeated cycles in the same woman). A GEE model with binomial distribution and a logit link function was used for the binary dependent variables (agonist protocol, the number of normal oocytes and the number of each abnormality on all the oocytes, clinical pregnancy rate, live birth rate, miscarriage rate), and a GEE model with Poisson distribution and a logit link function was used for count outcomes (the total number of retrieved oocytes, the number of matured oocytes, number of day 2-3 embryos obtained, number of frozen embryos and the number of fresh transferred embryos, cumulative CPR per cycle); and for ratios (proportion of MII oocytes, fertilization rate, grade 1 embryos rate, grade 3 embryos rate, implantation rate, cumulative CPR per transfer) by considering the numerators as dependent variable and the denominators as an offset variable. A linear mixed model with the patient as a random effect was used for comparisons of quantitative dependent variables (age of women, number of stimulation days, total dose of FSH, Estradiol on hCG day, AOQI score per cycle, average MOMS score per cycle). Comparisons of cycle outcomes were further adjusted for well-established, pre-specified confounding factors (namely the woman's age, BMI, smoking status, COH, and total dose of FSH). When analyzing the CPR for fresh embryos transfers, the number of fresh embryos transferred was also considered as a confounding factor. For the two cumulative CPRs (per cycle, and per total number of transfers), the number of total embryos transferred was considered as additional confounding factor. All statistical analyses were performed with SAS software (version 9.4, SAS Institute Inc., Cary, NC, USA). All tests were two-tailed, and the threshold for statistical significance was set to *p* < 0.05.

## Results

### The study population

A total of 109 women (aged between 22 and 37) were diagnosed with PCOS and had undergone a total of 189 ICSI cycles.
## Evaluation of embryos and ICSI cycles

Each MII oocyte was microinjected as part of a ICSI procedure described previously [38]. Fertilization (the presence of two distinct pronuclei) was scored 16-18 h after injection. Morphological embryo quality (i.e. the number of blastomeres, cell symmetry and fragmentation) was evaluated on day 2 or 3 according to the European Society of Human Reproduction and Embryology classification [35] A “top-quality embryo” was defined as the presence of four blastomeres on day 2 or eight blastomeres on day 3, symmetrical cleavage, and a fragmentation rate below 10% [35]. Top-quality embryos were scored as grade 1, and poor-quality embryos were scored as grade 3. Supernumerary top-quality embryos were frozen for subsequent transfer. Ultrasound-guided embryo transfer was performed 2 or 3 days after oocyte retrieval.

Luteal phase support (vaginal micronized progesterone 200 mg, 3 times a day) was indicated for all patients and was initiated on the evening of oocyte retrieval. The small proportion of freeze-all cycles (no fresh embryo transfers) was not taken into account in the comparisons of the ICSI outcomes.

## Outcomes

For the PCOS, PCOM-only and control groups, oocyte quality and morphology were assessed at the time of ICSI injection with regard to the oocyte maturity index (defined as the ratio between the number of MII oocytes and the total number of oocytes collected), the seven extracytoplasmic and intracytoplasmic abnormalities mentioned above, the AOQI, and the MOMS were performed.

After excluding cycles using frozen-thawed sperm, we evaluated the fertilization rate (FR), implantation rate (IR), clinical pregnancy rate (CPR), live birth rate (LBR) per transfer for day 2-3 embryos in fresh cycles, miscarriage rate (MR), and cumulative CPR. The IR was defined as the ratio between the number of gestational sacs with fetal heart activity and the number of embryos transferred. The live birth rate was defined as the number of live births per embryo transfer in fresh cycles. The cumulative CPR pregnancy rate was defined as the number of pregnancies with at least one gestational sac exhibiting fetal heart activity, including embryo transfers in fresh cycles and frozen-thawed cycles performed between 2006 and 2016.

## Statistical analysis

Quantitative variables were expressed as the mean ± standard deviation (for normally distributed datasets) or as the median [interquartile range (IQR)] otherwise. Normality of distributions was checked graphically and using Shapiro Wilk test. Categorical

variables were expressed by frequencies and percentages. Comparisons of subject's characteristics (at the first cycle) between the three PCOS phenotypes were performed with Chi-square test for current smoker women; and with analysis of variance for normally distributed quantitative variables or Kruskal-Wallis test otherwise. Post-hoc pairwise comparisons were performed in case of global significant difference and a Bonferroni correction was applied.

Groups were compared (i.e. PCOS vs PCOM-only vs controls; PCOS phenotypes A vs. C vs. D) using generalized estimating equation (GEE) models (to account for the correlation between repeated cycles in the same woman). A GEE model with binomial distribution and a logit link function was used for the binary dependent variables (agonist protocol, the number of normal oocytes and the number of each abnormality on all the oocytes, clinical pregnancy rate, live birth rate, miscarriage rate), and a GEE model with Poisson distribution and a logit link function was used for count outcomes (the total number of retrieved oocytes, the number of matured oocytes, number of day 2-3 embryos obtained, number of frozen embryos and the number of fresh transferred embryos, cumulative CPR per cycle); and for ratios (proportion of MII oocytes, fertilization rate, grade 1 embryos rate, grade 3 embryos rate, implantation rate, cumulative CPR per transfer) by considering the numerators as dependent variable and the denominators as an offset variable. A linear mixed model with the patient as a random effect was used for comparisons of quantitative dependent variables (age of women, number of stimulation days, total dose of FSH, Estradiol on hCG day, AOQI score per cycle, average MOMS score per cycle). Comparisons of cycle outcomes were further adjusted for well-established, pre-specified confounding factors (namely the woman's age, BMI, smoking status, COH, and total dose of FSH). When analyzing the CPR for fresh embryos transfers, the number of fresh embryos transferred was also considered as a confounding factor. For the two cumulative CPRs (per cycle, and per total number of transfers), the number of total embryos transferred was considered as additional confounding factor. All statistical analyses were performed with SAS software (version 9.4, SAS Institute Inc., Cary, NC, USA). All tests were two-tailed, and the threshold for statistical significance was set to *p* < 0.05.

## Results

### The study population

A total of 109 women (aged between 22 and 37) were diagnosed with PCOS and had undergone a total of 189 ICSI cycles.
<table><caption>**Table 3** Clinical and endocrine characteristics, by PCOS phenotype</caption><thead><tr><th>PCOS B (n = 1) was excluded</th><th>PCOS A<br>N = 41</th><th>PCOS C<br>N = 31</th><th>PCOS D<br>N = 37</th><th>p value</th></tr></thead><tbody><tr><td>Woman's age at first ICSI cycle (y)</td><td>29.2 ± 3.2</td><td>28.6 ± 4.1</td><td>29.1 ± 3.4</td><td>0.77</td></tr><tr><td>Man's age (y)</td><td>32.9 ± 4.5</td><td>32.0 ± 7.2</td><td>32.6 ± 4.5</td><td>0.80</td></tr><tr><td>Current smoking, n (%)</td><td>9 (22.0)</td><td>14 (45.2) <sup>c</sup></td><td>5 (13.5) <sup>b</sup></td><td>0.009</td></tr><tr><td>BMI (kg/m<sup>2</sup>)</td><td>27.7 ± 6.7</td><td>26.4 ± 6.0</td><td>26.3 ± 5.0</td><td>0.48</td></tr><tr><td>Waist circumference (cm)</td><td>86.6 ± 16.3</td><td>84.0 ± 14.8</td><td>84.6 ± 12.8</td><td>0.74</td></tr><tr><td>Testosterone (ng/mL)</td><td>0.4 ± 0.2<sup>c</sup></td><td>0.3 ± 0.2</td><td>0.3 ± 0.1<sup>a</sup></td><td>0.004</td></tr><tr><td>Delta-4-androstenedione (ng/mL)</td><td>2.3 ± 0.7<sup>c</sup></td><td>2.1 ± 0.7<sup>c</sup></td><td>1.3 ± 0.4<sup>ab</sup></td><td>&lt;0.001</td></tr><tr><td>LH (IU/L)</td><td>5.6 ± 2.6<sup>bc</sup></td><td>4.1 ± 1.9<sup>a</sup></td><td>4.2 ± 2.0<sup>a</sup></td><td>0.006</td></tr><tr><td>FSH (IU/L)</td><td>4.8 ± 1.1</td><td>5.2 ± 1.4</td><td>4.7 ± 1.0</td><td>0.23</td></tr><tr><td>LH/FSH ratio (IU/L)</td><td>1.1 [0.9-1.4]<sup>bc</sup></td><td>0.8 [0.6-1.0]<sup>a</sup></td><td>0.8 [0.7-1.2]<sup>a</sup></td><td>&lt;0.001</td></tr><tr><td>AMH (pmol/L)</td><td>74.8 ± 37.4<sup>bc</sup></td><td>47.3 ± 17.1<sup>a</sup></td><td>56.2 ± 19.9<sup>a</sup></td><td>&lt;0.001</td></tr><tr><td>FNPO (n)</td><td>26.8 ± 10.1</td><td>21.7 ± 8.2</td><td>23.1 ± 8.0</td><td>0.042</td></tr></tbody></table>

Values are quoted as the mean ± standard deviation, the mean [IQR], or n (%)

Abbreviations: y years, BMI body mass index, LH luteinizing hormone, FSH follicle-stimulating hormone, IQR interquartile range, AMH anti-Müllerian hormone, FNPO follicle number per ovary

The clinical and endocrine characteristics of the phenotype groups are summarized in Table 3. There were no statistically significant differences between the three phenotype groups with regard to the woman's age at the time of the first ICSI attempt, the BMI, the male partner's age, or the FSH levels. The serum AMH level was significantly higher for phenotype A (74.8 ± 37.4) than for phenotypes C (47.3 ± 17.1) and D (56.2 ± 19.9) (*p* < 0.001). Androstenedione levels were significantly higher for phenotypes A and C than for phenotype D. The only

significant difference in the serum testosterone concentration concerned phenotypes A and D (respectively, 0.4 ± 0.2 vs 0.3 ± 0.1; *p* = 0.004). The characteristics of the COH and outcomes for respectively 77, 57 and 55 cycles in the phenotype A, C and D groups are summarized in Table 4. There were no significant intergroup differences in the proportion of agonist protocols, the total dose of gonadotropin, and the total numbers of retrieved and MII oocytes.

<table><caption>**Table 4** Clinical and laboratory characteristics in ICSI cycles, by PCOS phenotype</caption><thead><tr><th>PCOS B (n = 1) was excluded</th><th>PCOS A<br>N = 41</th><th>PCOS C<br>N = 31</th><th>PCOS D<br>N = 37</th><th>p value</th><th>Adjusted p</th></tr></thead><tbody><tr><td>Number of ICSI cycles</td><td>77</td><td>57</td><td>55</td><td>—</td><td>—</td></tr><tr><td>Woman's age during the ICSI cycle</td><td>29.6 ± 3.3</td><td>29.0 ± 4.3</td><td>29.7 ± 3.2</td><td>0.48</td><td>—</td></tr><tr><td>Protocol:</td><td></td><td></td><td></td><td>0.73</td><td>—</td></tr><tr><td>Agonist, n (%)</td><td>38 (49.4)</td><td>29 (50.9)</td><td>24 (43.6)</td><td>—</td><td>—</td></tr><tr><td>Antagonist, n (%)</td><td>39 (50.6)</td><td>28 (49.1)</td><td>31 (56.4)</td><td>—</td><td>—</td></tr><tr><td>Number of days of stimulation</td><td>12.9 ± 2.5</td><td>12.1 ± 2.5</td><td>12.1 ± 1.9</td><td>0.15</td><td>—</td></tr><tr><td>Total dose of FSH, IU</td><td>1812 ± 872</td><td>2016 ± 1076</td><td>1741 ± 700</td><td>0.41</td><td>—</td></tr><tr><td>Estradiol on hCG day, pg/mL</td><td>2666 ± 1454</td><td>2363 ± 1387</td><td>2382 ± 1206</td><td>0.42</td><td>—</td></tr><tr><td>Mean number of oocytes retrieved (N)</td><td>12.2 ± 6.0</td><td>12.1 ± 5.2</td><td>11.8 ± 6.2</td><td>0.92</td><td>0.92</td></tr><tr><td>Mean number of MII oocytes (n)</td><td>7.8 ± 4.6</td><td>8.1 ± 3.6</td><td>7.9 ± 4.8</td><td>0.91</td><td>0.98</td></tr><tr><td>Proportion of MII oocytes, n/N (%)</td><td>602/939 (64.1)</td><td>462/692 (66.8)</td><td>432/648 (66.7)</td><td>0.85</td><td>0.94</td></tr></tbody></table>

Values are quoted as the mean ± standard deviation or n (%)

The *p* value was adjusted for the woman's age, BMI and current smoking status, the type of COH protocol, and the total dose of FSH

Abbreviations: FSH follicle-stimulating hormone, MII metaphase II

<footnote><sup>a</sup> *p* < 0.05 vs. PCOS A after Bonferroni correction</footnote>

<footnote><sup>b</sup> *p* < 0.05 vs. PCOS C after Bonferroni correction</footnote>

<footnote><sup>c</sup> *p* < 0.05 vs. PCOS D after Bonferroni correction</footnote>
<table><caption>**Table 3** Clinical and endocrine characteristics, by PCOS phenotype</caption><thead><tr><th>PCOS B (n = 1) was excluded</th><th>PCOS A<br>N = 41</th><th>PCOS C<br>N = 31</th><th>PCOS D<br>N = 37</th><th>p value</th></tr></thead><tbody><tr><td>Woman's age at first ICSI cycle (y)</td><td>29.2 ± 3.2</td><td>28.6 ± 4.1</td><td>29.1 ± 3.4</td><td>0.77</td></tr><tr><td>Man's age (y)</td><td>32.9 ± 4.5</td><td>32.0 ± 7.2</td><td>32.6 ± 4.5</td><td>0.80</td></tr><tr><td>Current smoking, n (%)</td><td>9 (22.0)</td><td>14 (45.2) <sup>c</sup></td><td>5 (13.5) <sup>b</sup></td><td>0.009</td></tr><tr><td>BMI (kg/m<sup>2</sup>)</td><td>27.7 ± 6.7</td><td>26.4 ± 6.0</td><td>26.3 ± 5.0</td><td>0.48</td></tr><tr><td>Waist circumference (cm)</td><td>86.6 ± 16.3</td><td>84.0 ± 14.8</td><td>84.6 ± 12.8</td><td>0.74</td></tr><tr><td>Testosterone (ng/mL)</td><td>0.4 ± 0.2<sup>c</sup></td><td>0.3 ± 0.2</td><td>0.3 ± 0.1<sup>a</sup></td><td>0.004</td></tr><tr><td>Delta-4-androstenedione (ng/mL)</td><td>2.3 ± 0.7<sup>c</sup></td><td>2.1 ± 0.7<sup>c</sup></td><td>1.3 ± 0.4<sup>ab</sup></td><td>&lt;0.001</td></tr><tr><td>LH (IU/L)</td><td>5.6 ± 2.6<sup>bc</sup></td><td>4.1 ± 1.9<sup>a</sup></td><td>4.2 ± 2.0<sup>a</sup></td><td>0.006</td></tr><tr><td>FSH (IU/L)</td><td>4.8 ± 1.1</td><td>5.2 ± 1.4</td><td>4.7 ± 1.0</td><td>0.23</td></tr><tr><td>LH/FSH ratio (IU/L)</td><td>1.1 [0.9-1.4]<sup>bc</sup></td><td>0.8 [0.6-1.0]<sup>a</sup></td><td>0.8 [0.7-1.2]<sup>a</sup></td><td>&lt;0.001</td></tr><tr><td>AMH (pmol/L)</td><td>74.8 ± 37.4<sup>bc</sup></td><td>47.3 ± 17.1<sup>a</sup></td><td>56.2 ± 19.9<sup>a</sup></td><td>&lt;0.001</td></tr><tr><td>FNPO (n)</td><td>26.8 ± 10.1</td><td>21.7 ± 8.2</td><td>23.1 ± 8.0</td><td>0.042</td></tr></tbody></table>

Values are quoted as the mean ± standard deviation, the mean [IQR], or n (%)

Abbreviations: y years, BMI body mass index, LH luteinizing hormone, FSH follicle-stimulating hormone, IQR interquartile range, AMH anti-Müllerian hormone, FNPO follicle number per ovary

The clinical and endocrine characteristics of the phenotype groups are summarized in Table 3. There were no statistically significant differences between the three phenotype groups with regard to the woman's age at the time of the first ICSI attempt, the BMI, the male partner's age, or the FSH levels. The serum AMH level was significantly higher for phenotype A (74.8 ± 37.4) than for phenotypes C (47.3 ± 17.1) and D (56.2 ± 19.9) (*p* < 0.001). Androstenedione levels were significantly higher for phenotypes A and C than for phenotype D. The only

significant difference in the serum testosterone concentration concerned phenotypes A and D (respectively, 0.4 ± 0.2 vs 0.3 ± 0.1; *p* = 0.004). The characteristics of the COH and outcomes for respectively 77, 57 and 55 cycles in the phenotype A, C and D groups are summarized in Table 4. There were no significant intergroup differences in the proportion of agonist protocols, the total dose of gonadotropin, and the total numbers of retrieved and MII oocytes.

<table><caption>**Table 4** Clinical and laboratory characteristics in ICSI cycles, by PCOS phenotype</caption><thead><tr><th>PCOS B (n = 1) was excluded</th><th>PCOS A<br>N = 41</th><th>PCOS C<br>N = 31</th><th>PCOS D<br>N = 37</th><th>p value</th><th>Adjusted p</th></tr></thead><tbody><tr><td>Number of ICSI cycles</td><td>77</td><td>57</td><td>55</td><td>—</td><td>—</td></tr><tr><td>Woman's age during the ICSI cycle</td><td>29.6 ± 3.3</td><td>29.0 ± 4.3</td><td>29.7 ± 3.2</td><td>0.48</td><td>—</td></tr><tr><td>Protocol:</td><td></td><td></td><td></td><td>0.73</td><td>—</td></tr><tr><td>Agonist, n (%)</td><td>38 (49.4)</td><td>29 (50.9)</td><td>24 (43.6)</td><td>—</td><td>—</td></tr><tr><td>Antagonist, n (%)</td><td>39 (50.6)</td><td>28 (49.1)</td><td>31 (56.4)</td><td>—</td><td>—</td></tr><tr><td>Number of days of stimulation</td><td>12.9 ± 2.5</td><td>12.1 ± 2.5</td><td>12.1 ± 1.9</td><td>0.15</td><td>—</td></tr><tr><td>Total dose of FSH, IU</td><td>1812 ± 872</td><td>2016 ± 1076</td><td>1741 ± 700</td><td>0.41</td><td>—</td></tr><tr><td>Estradiol on hCG day, pg/mL</td><td>2666 ± 1454</td><td>2363 ± 1387</td><td>2382 ± 1206</td><td>0.42</td><td>—</td></tr><tr><td>Mean number of oocytes retrieved (N)</td><td>12.2 ± 6.0</td><td>12.1 ± 5.2</td><td>11.8 ± 6.2</td><td>0.92</td><td>0.92</td></tr><tr><td>Mean number of MII oocytes (n)</td><td>7.8 ± 4.6</td><td>8.1 ± 3.6</td><td>7.9 ± 4.8</td><td>0.91</td><td>0.98</td></tr><tr><td>Proportion of MII oocytes, n/N (%)</td><td>602/939 (64.1)</td><td>462/692 (66.8)</td><td>432/648 (66.7)</td><td>0.85</td><td>0.94</td></tr></tbody></table>

Values are quoted as the mean ± standard deviation or n (%)

The *p* value was adjusted for the woman's age, BMI and current smoking status, the type of COH protocol, and the total dose of FSH

Abbreviations: FSH follicle-stimulating hormone, MII metaphase II

<footnote><sup>a</sup> *p* < 0.05 vs. PCOS A after Bonferroni correction</footnote>

<footnote><sup>b</sup> *p* < 0.05 vs. PCOS C after Bonferroni correction</footnote>

<footnote><sup>c</sup> *p* < 0.05 vs. PCOS D after Bonferroni correction</footnote>
The clinical and endocrine characteristics of the PCOS, PCOM-only and control groups are summarized in supplemental Table 1. The BMI was significantly higher in the PCOS group than in the control group (p = 0.025). The waist circumference was significantly higher in the PCOS group than in the control group (85.0 ± 14.7 vs 78.5 ± 12.3, p = 0.012). The serum testosterone level and the serum delta-4 androstenedione level were significantly higher (p < 0.001 and < 0.001, respectively) in the PCOS group than in the PCOM-only and control groups. Furthermore, the LH/FSH ratio was significantly higher in the PCOS group than in the two other groups. The antral follicle count and serum AMH levels were significantly higher (i) in the PCOS group than in the PCOM-only and control groups, and (ii) in the PCOM-only group than in the control group (Supplemental Table 1). There were no significant intergroup differences in the woman's age, the duration of stimulation, and the serum estradiol level on the trigger day. The numbers of retrieved oocytes and MII oocytes were significantly higher in the PCOS and PCOM-only groups per cycle (respectively: 12.0 ± 5.7; 7.9 ± 4.3) and PCOM-only groups (respectively: 12.4 ± 6.2; 7.9 ± 4.2) than in control group (respectively: 9.5 ± 4.7; 6.2 ± 3.9). However, no difference was found in the mature oocyte rate between the three groups (supplemental Table 2).

## Oocyte morphology comparison

A total of 3312 oocytes were assessed morphologically: 1523 oocytes in the PCOS group; 1059 in the PCOM-only group; 730 in the control group.

After adjustment for the woman's age, BMI and smoking status and the total dose of FSH administered, a detailed analysis of each morphologic abnormality did not reveal any significant differences between the three PCOS phenotypes (Table 5). The percentages of normal oocytes were similar for PCOS phenotypes A, C and D (percentages (95%CI): 35.2% (31.5 to 39.1%), 25.8% (21.9 to 29.9%) and 34.0% (29.7 to 38.6%), adjusted *p* = 0.13). Similarly, there were no intergroup differences in oocyte morphologic quality (as expressed by the AOQI (means (95% CI)) for A, C and D respectively: 0.76 (0.68 to 0.85), 0.85 (0.72 to 0.98), 0.75 (0.64 to 0.86), adjusted *p* = 0.40) and the MOMS for A, C and D respectively: 1.31 (1.04-1.46), 1.30 (1.10-1.50), 1.27 (1.08-1.46), adjusted *p* = 0.83)).

Lastly, there were no differences between the PCOS, PCOM-only and control groups in the normal oocyte rate (adjusted percentages (95%CI): 31.8% (29.5 to 34.2%), 30.6% (27.9 to 33.4%), 27.8% (24.7 to 31.2%) respectively, adjusted *p* = 0.90), the AOQI or the MOMS (Supplemental Table 3).

<table><caption>**Table 5** Oocyte morphologic quality, by PCOS phenotype</caption><thead><tr><th></th><th>PCOS A<br>N = 41</th><th>PCOS C<br>N = 31</th><th>PCOS D<br>N = 37</th><th>p value</th><th>Adjusted p</th></tr></thead><tbody><tr><td>Number of MII oocytes</td><td>602</td><td>462</td><td>432</td><td>–</td><td>–</td></tr><tr><td>Normal oocytes, n (%)</td><td>212<br>(35.2 [31.5 to 39.1])</td><td>119<br>(25.8 [21.9 to 29.9])</td><td>147<br>(34.0 [29.7 to 38.6])</td><td>0.031</td><td>0.13</td></tr><tr><td>Fragmented or abnormal FPB, n (%)</td><td>299<br>(49.7 [45.7 to 53.7])</td><td>245<br>(53.0 [48.5 to 57.5])</td><td>241<br>(55.8 [51.1 to 60.4])</td><td>0.16</td><td>0.15</td></tr><tr><td>Abnormal zona pellucida, n (%)</td><td>8<br>(1.3 [0.7 to 2.6])</td><td>6<br>(1.3 [0.6 to 2.9])</td><td>3<br>(0.7 [0.2 to 2.1])</td><td>0.60</td><td>0.75</td></tr><tr><td>Large perivitelline space, n (%)</td><td>49<br>(8.1 [6.2 to 10.6])</td><td>56<br>(12.1 [9.4 to 15.4])</td><td>29<br>(6.7 [4.7 to 9.5])</td><td>0.36</td><td>0.34</td></tr><tr><td>Material in perivitelline space, n (%)</td><td>68<br>(11.3 [9.0 to 14.1])</td><td>52<br>(11.3 [8.9 to 14.5])</td><td>41<br>(9.5 [7.1 to 12.6])</td><td>0.89</td><td>0.80</td></tr><tr><td>Abnormal shape of oocyte, n (%)</td><td>19<br>(3.2 [2.0 to 4.9])</td><td>14<br>(3.0 [1.8 to 5.1])</td><td>9<br>(2.1 [1.1 to 4.0])</td><td>0.41</td><td>0.39</td></tr><tr><td>Granular cytoplasm, n (%)</td><td>21<br>(3.5 [2.3 to 5.3])</td><td>16<br>(3.5 [2.1 to 5.6])</td><td>16<br>(3.7 [2.3 to 6.0])</td><td>0.92</td><td>0.81</td></tr><tr><td>Intracytoplasmic vacuoles, n (%)</td><td>12<br>(2.0 [1.1 to 3.5])</td><td>15<br>(3.3 [2.0 to 5.3])</td><td>3<br>(0.7 [0.2 to 2.1])</td><td>0.049</td><td>0.053</td></tr><tr><td>AOQI, mean ± SD<sup>a</sup></td><td>0.76 [0.68 to 0.85]</td><td>0.85 [0.72 to 0.98]</td><td>0.75 [0.64 to 0.86]</td><td>0.29</td><td>0.40</td></tr><tr><td>average MOMS per cycle, mean ± SD<sup>a</sup></td><td>1.31 [1.04 to 1.46]</td><td>1.30 [1.10 to 1.50]</td><td>1.27 [1.08 to 1.46]</td><td>0.80</td><td>0.83</td></tr></tbody></table>

Values are quoted as the mean [95%CI] or n (% [95%CI]). The *p* value was adjusted for the woman's age, BMI and current smoking status, the type of COH protocol, and the total dose of FSH

Abbreviations: MII metaphase II, FPB first polar body, ZP zona pellucida, AOQI average oocyte quality index, MOMS metaphase II oocyte morphologic score

<footnote>ª The AOQI score considers all the oocyte morphologic abnormalities per cycle (Sigala et al., 2015 [36]), while the MOMS takes account of the morphologic abnormalities per oocyte. However, the MOMS was averaged by cycle here, in order to compare the groups (Rienzi et al., 2008 [37])</footnote>
The clinical and endocrine characteristics of the PCOS, PCOM-only and control groups are summarized in supplemental Table 1. The BMI was significantly higher in the PCOS group than in the control group ($p=0.025$). The waist circumference was significantly higher in the PCOS group than in the control group ($85.0 \pm 14.7$ vs $78.5 \pm 12.3$, $p=0.012$). The serum testosterone level and the serum delta-4 androstenedione level were significantly higher ($p<0.001$ and $<0.001$, respectively) in the PCOS group than in the PCOM-only and control groups. Furthermore, the LH/FSH ratio was significantly higher in the PCOS group than in the two other groups. The antral follicle count and serum AMH levels were significantly higher (i) in the PCOS group than in the PCOM-only and control groups, and (ii) in the PCOM-only group than in the control group (Supplemental Table 1). There were no significant intergroup differences in the woman's age, the duration of stimulation, and the serum estradiol level on the trigger day. The numbers of retrieved oocytes and MII oocytes were significantly higher in the PCOS and PCOM-only groups per cycle (respectively: $12.0 \pm 5.7$; $7.9 \pm 4.3$) and PCOM-only groups (respectively: $12.4 \pm 6.2$; $7.9 \pm 4.2$) than in control group (respectively: $9.5 \pm 4.7$; $6.2 \pm 3.9$). However, no difference was found in the mature oocyte rate between the three groups (supplemental Table 2).

## Oocyte morphology comparison

A total of 3312 oocytes were assessed morphologically: 1523 oocytes in the PCOS group; 1059 in the PCOM-only group; 730 in the control group.

After adjustment for the woman's age, BMI and smoking status and the total dose of FSH administered, a detailed analysis of each morphologic abnormality did not reveal any significant differences between the three PCOS phenotypes (Table 5). The percentages of normal oocytes were similar for PCOS phenotypes A, C and D (percentages (95%CI): 35.2% (31.5 to 39.1%), 25.8% (21.9 to 29.9%) and 34.0% (29.7 to 38.6%), adjusted $p=0.13$). Similarly, there were no intergroup differences in oocyte morphologic quality (as expressed by the AOQI (means (95% CI)) for A, C and D respectively: 0.76 (0.68 to 0.85), 0.85 (0.72 to 0.98), 0.75 (0.64 to 0.86), adjusted $p=0.40$) and the MOMS for A, C and D respectively: 1.31 (1.04-1.46), 1.30 (1.10-1.50), 1.27 (1.08-1.46), adjusted $p=0.83$)).

Lastly, there were no differences between the PCOS, PCOM-only and control groups in the normal oocyte rate (adjusted percentages (95%CI): 31.8% (29.5 to 34.2%), 30.6% (27.9 to 33.4%), 27.8% (24.7 to 31.2%) respectively, adjusted $p=0.90$), the AOQI or the MOMS (Supplemental Table 3).

<table><caption>**Table 5** Oocyte morphologic quality, by PCOS phenotype</caption><thead><tr><th></th><th>PCOS A<br>N=41</th><th>PCOS C<br>N=31</th><th>PCOS D<br>N=37</th><th>p value</th><th>Adjusted p</th></tr></thead><tbody><tr><td>Number of MII oocytes</td><td>602</td><td>462</td><td>432</td><td>–</td><td>–</td></tr><tr><td>Normal oocytes, n (%)</td><td>212<br>(35.2 [31.5 to 39.1])</td><td>119<br>(25.8 [21.9 to 29.9])</td><td>147<br>(34.0 [29.7 to 38.6])</td><td>0.031</td><td>0.13</td></tr><tr><td>Fragmented or abnormal FPB, n (%)</td><td>299<br>(49.7 [45.7 to 53.7])</td><td>245<br>(53.0 [48.5 to 57.5])</td><td>241<br>(55.8 [51.1 to 60.4])</td><td>0.16</td><td>0.15</td></tr><tr><td>Abnormal zona pellucida, n (%)</td><td>8<br>(1.3 [0.7 to 2.6])</td><td>6<br>(1.3 [0.6 to 2.9])</td><td>3<br>(0.7 [0.2 to 2.1])</td><td>0.60</td><td>0.75</td></tr><tr><td>Large perivitelline space, n (%)</td><td>49<br>(8.1 [6.2 to 10.6])</td><td>56<br>(12.1 [9.4 to 15.4])</td><td>29<br>(6.7 [4.7 to 9.5])</td><td>0.36</td><td>0.34</td></tr><tr><td>Material in perivitelline space, n (%)</td><td>68<br>(11.3 [9.0 to 14.1])</td><td>52<br>(11.3 [8.9 to 14.5])</td><td>41<br>(9.5 [7.1 to 12.6])</td><td>0.89</td><td>0.80</td></tr><tr><td>Abnormal shape of oocyte, n (%)</td><td>19<br>(3.2 [2.0 to 4.9])</td><td>14<br>(3.0 [1.8 to 5.1])</td><td>9<br>(2.1 [1.1 to 4.0])</td><td>0.41</td><td>0.39</td></tr><tr><td>Granular cytoplasm, n (%)</td><td>21<br>(3.5 [2.3 to 5.3])</td><td>16<br>(3.5 [2.1 to 5.6])</td><td>16<br>(3.7 [2.3 to 6.0])</td><td>0.92</td><td>0.81</td></tr><tr><td>Intracytoplasmic vacuoles, n (%)</td><td>12<br>(2.0 [1.1 to 3.5])</td><td>15<br>(3.3 [2.0 to 5.3])</td><td>3<br>(0.7 [0.2 to 2.1])</td><td>0.049</td><td>0.053</td></tr><tr><td>AOQI, mean ± SD<sup>a</sup></td><td>0.76 [0.68 to 0.85]</td><td>0.85 [0.72 to 0.98]</td><td>0.75 [0.64 to 0.86]</td><td>0.29</td><td>0.40</td></tr><tr><td>average MOMS per cycle, mean ± SD<sup>a</sup></td><td>1.31 [1.04 to 1.46]</td><td>1.30 [1.10 to 1.50]</td><td>1.27 [1.08 to 1.46]</td><td>0.80</td><td>0.83</td></tr></tbody></table>

Values are quoted as the mean [95%CI] or n (% [95%CI]). The *p* value was adjusted for the woman's age, BMI and current smoking status, the type of COH protocol, and the total dose of FSH

Abbreviations: MII metaphase II, FPB first polar body, ZP zona pellucida, AOQI average oocyte quality index, MOMS metaphase II oocyte morphologic score

<footnote><sup>a</sup> The AOQI score considers all the oocyte morphologic abnormalities per cycle (Sigala et al., 2015 [36]), while the MOMS takes account of the morphologic abnormalities per oocyte. However, the MOMS was averaged by cycle here, in order to compare the groups (Rienzi et al., 2008 [37])</footnote>
<table><caption>**Table 6** Embryo morphologic quality and ICSI outcomes, by PCOS phenotype</caption><thead><tr><th></th><th>PCOS A</th><th>PCOS C</th><th>PCOS D</th><th>p value</th><th>Adjusted p</th></tr></thead><tbody><tr><td>Number of ICSI cycles</td><td>70</td><td>48</td><td>42</td><td>—</td><td>—</td></tr><tr><td>Fertilization rate, n/n<sub>MIII ovo</sub> (%)</td><td>331/536 (61.8 [55.4 to 68.8])</td><td>233/394 (59.1 [52.0 to 67.2])</td><td>205/346 (59.2 [51.7 to 67.9])</td><td>0.93</td><td>0.93</td></tr><tr><td>Number of day 2-3 embryos</td><td>4.6 [4.1 to 5.1]</td><td>4.6 [4.1 to 5.3]</td><td>4.8 [4.2 to 5.5]</td><td>0.92</td><td>0.84</td></tr><tr><td>Grade 1 embryos (%)<sup>‡</sup></td><td>159/319 (49.8 [42.7 to 58.2])</td><td>113/222 (50.9 [42.3 to 61.2])</td><td>104/201 (51.7 [42.7 to 62.7])</td><td>0.93</td><td>0.86</td></tr><tr><td>Grade 3 embryos (%)<sup>‡</sup></td><td>130/319 (40.8 [34.3 to 48.4])</td><td>89/222 (40.1 [32.6 to 46.8])</td><td>75/201 (37.3 [29.8 to 46.8])</td><td>0.76</td><td>0.58</td></tr><tr><td>Number of frozen embryos (n) <sup>‡</sup></td><td>1.7 [1.4 to 2.0]</td><td>1.5 [1.2 to 1.9]</td><td>2.0 [1.6 to 2.5]</td><td>0.59</td><td>0.57</td></tr><tr><td>Fresh transferred embryos (n)<sup>‡</sup></td><td>1.4 [1.1 to 1.7]</td><td>1.6 [1.3 to 2.0]</td><td>1.4 [1.1 to 1.8]</td><td>0.42</td><td>0.39</td></tr><tr><td>Implantation rate, n/n<sub>embryos transferred</sub> (%)*</td><td>28/89 (31.5 [21.7 to 45.6])</td><td>21/73 (28.8 [18.8 to 44.1])</td><td>18/56 (32.1 [20.3 to 51.0])</td><td>0.79</td><td>0.82</td></tr><tr><td>CPR, n/n<sub>transfers with fresh embryos</sub> (%)*</td><td>26/53 (49.1 [36.0 to 62.3])</td><td>20/39 (51.3 [36.0 to 66.4])</td><td>15/33 (45.5 [29.6 to 62.3])</td><td>0.88</td><td>0.71<sup>f</sup></td></tr><tr><td>Live birth rate, n/n<sub>transfers with fresh embryos</sub> (%)*</td><td>19/53 (35.8 [24.2 to 49.5])</td><td>16/39 (41.0 [26.9 to 56.8])</td><td>13/33 (39.4 [24.4 to 56.6])</td><td>0.92</td><td>0.44</td></tr><tr><td>Miscarriage rate, n/n<sub>clinical pregnancy</sub> (%)*</td><td>5/26 (19.2 [8.2 to 38.7])</td><td>3/20 (15.0 [4.9 to 37.6])</td><td>2/15 (13.3 [3.4 to 40.5])</td><td>0.89</td><td>NA</td></tr><tr><td>Cumulative CPR, n/n<sub>total transfers</sub> (%)</td><td>33/94 (35.1 [25.0 to 49.4])</td><td>26/65 (40.0 [27.2 to 58.8])</td><td>29/68 (42.6 [28.4 to 59.6])</td><td>0.51</td><td>0.43<sup>#</sup></td></tr><tr><td>Cumulative CPR, n/n<sub>cycles</sub> (%)</td><td>33/70 (47.1 [33.5 to 66.3])</td><td>26/48 (54.2 [36.9 to 79.6])</td><td>29/42 (69.0 [48.0 to 99.4])</td><td>0.15</td><td>0.18<sup>#</sup></td></tr></tbody></table>

Values are quoted as the mean [95% CI] or no./total no. (rate [95%CI] in %). The *p* value was adjusted for the woman's age, BMI and current smoking status, the type of COH protocol, and the total dose of rFSH

<sup>f</sup> additionally adjusted for the number of fresh embryos transferred

<sup>#</sup> additionally adjusted for the total number of embryos transferred

Abbreviations: CPR clinical pregnancy rate, NA not applicable, due to low frequencies

<sup>‡</sup> cycles with no embryos obtained on day 2-3 were excluded

* cycles with embryo transfer on day 5-6 and cycles with a “freeze-all” strategy were excluded

## ICSI outcomes

After the exclusion of ICSI cycles requiring frozen-thawed sperm, the ICSI outcomes are shown in Table 6. There was no significant phenotype-related difference in the fertilization rate (percentages [95%CI] for A, C and D respectively: 61.8% (55.4 to 68.8%), 59.1% (52.0 to 67.2%), 59.2% (51.7 to 67.9%), adjusted *p*=0.93) or the mean number of day 2-3 embryos obtained (mean [95% CI]) for A, C and D respectively: 4.6 (4.1-5.1), 4.6 (4.1-5.3), 4.8 (4.2-5.5), *p*=0.84). The percentages of good quality (grade 1) embryos (percentage [95%CI] for A, C and D respectively: 49.8% (42.7 to 58.2%), 50.9% (42.3 to 61.2%), 51.7% (42.7 to 62.7%), adjusted *p*=0.86) and poor quality (grade 3) embryos (percentage [95%CI] for A, C and D respectively: 40.8% (34.3 to 48.4%), 40.1% (32.5 to 49.4%), 37.3% (29.7 to 46.8%), adjusted *p*=0.58) were also similar in the three phenotype groups. After adjustment for confounding factors we did not detect any intergroup differences in the IR, CPR, LBR, MR, and cumulative CPR. With regard

to ICSI outcomes, only the number of D2-D3 embryos and the number of frozen embryos were significantly higher in the PCOS group than in the PCOM and control groups (Supplemental Table 4).

## Discussion

Although a few published studies have investigated reproductive IVF/ICSI outcomes among women with PCOS, oocyte morphologic quality for the various PCOS phenotypes has not previously been described. Indeed, previous research by our group found that oocyte quality was similar in a PCOM group and a control women; however, the study's PCOM group contained a mixture of PCOM-only women and PCOS women [36]. It is well established that PCOM is a distinct phenotype and is not the same as PCOS [31]. We therefore decided to investigate oocyte morphology among the different PCOS phenotypes. To the best of our knowledge, the present study is the first to have addressed this topic. Our study population was homogenous and met strict inclusion
<table><caption>**Table 6** Embryo morphologic quality and ICSI outcomes, by PCOS phenotype</caption><thead><tr><th></th><th>PCOS A</th><th>PCOS C</th><th>PCOS D</th><th>p value</th><th>Adjusted p</th></tr></thead><tbody><tr><td>Number of ICSI cycles</td><td>70</td><td>48</td><td>42</td><td>-</td><td>-</td></tr><tr><td>Fertilization rate, n/n<sub>MIII ovo</sub> (%)</td><td>331/536 (61.8 [55.4 to 68.8])</td><td>233/394 (59.1 [52.0 to 67.2])</td><td>205/346 (59.2 [51.7 to 67.9])</td><td>0.93</td><td>0.93</td></tr><tr><td>Number of day 2-3 embryos</td><td>4.6 [4.1 to 5.1]</td><td>4.6 [4.1 to 5.3]</td><td>4.8 [4.2 to 5.5]</td><td>0.92</td><td>0.84</td></tr><tr><td>Grade 1 embryos (%)<sup>‡</sup></td><td>159/319 (49.8 [42.7 to 58.2])</td><td>113/222 (50.9 [42.3 to 61.2])</td><td>104/201 (51.7 [42.7 to 62.7])</td><td>0.93</td><td>0.86</td></tr><tr><td>Grade 3 embryos (%)<sup>‡</sup></td><td>130/319 (40.8 [34.3 to 48.4])</td><td>89/222 (40.1 [32.6 to 46.8])</td><td>75/201 (37.3 [29.8 to 46.8])</td><td>0.76</td><td>0.58</td></tr><tr><td>Number of frozen embryos (n) <sup>‡</sup></td><td>1.7 [1.4 to 2.0]</td><td>1.5 [1.2 to 1.9]</td><td>2.0 [1.6 to 2.5]</td><td>0.59</td><td>0.57</td></tr><tr><td>Fresh transferred embryos (n)<sup>‡</sup></td><td>1.4 [1.1 to 1.7]</td><td>1.6 [1.3 to 2.0]</td><td>1.4 [1.1 to 1.8]</td><td>0.42</td><td>0.39</td></tr><tr><td>Implantation rate, n/n<sub>embryos transferred</sub> (%)*</td><td>28/89 (31.5 [21.7 to 45.6])</td><td>21/73 (28.8 [18.8 to 44.1])</td><td>18/56 (32.1 [20.3 to 51.0])</td><td>0.79</td><td>0.82</td></tr><tr><td>CPR, n/n<sub>transfers with fresh embryos</sub> (%)*</td><td>26/53 (49.1 [36.0 to 62.3])</td><td>20/39 (51.3 [36.0 to 66.4])</td><td>15/33 (45.5 [29.6 to 62.3])</td><td>0.88</td><td>0.71<sup>f</sup></td></tr><tr><td>Live birth rate, n/n<sub>transfers with fresh embryos</sub> (%)*</td><td>19/53 (35.8 [24.2 to 49.5])</td><td>16/39 (41.0 [26.9 to 56.8])</td><td>13/33 (39.4 [24.4 to 56.6])</td><td>0.92</td><td>0.44</td></tr><tr><td>Miscarriage rate, n/n<sub>clinical pregnancy</sub> (%)*</td><td>5/26 (19.2 [8.2 to 38.7])</td><td>3/20 (15.0 [4.9 to 37.6])</td><td>2/15 (13.3 [3.4 to 40.5])</td><td>0.89</td><td>NA</td></tr><tr><td>Cumulative CPR, n/n<sub>total transfers</sub> (%)</td><td>33/94 (35.1 [25.0 to 49.4])</td><td>26/65 (40.0 [27.2 to 58.8])</td><td>29/68 (42.6 [28.4 to 59.6])</td><td>0.51</td><td>0.43<sup>#</sup></td></tr><tr><td>Cumulative CPR, n/n<sub>cycles</sub> (%)</td><td>33/70 (47.1 [33.5 to 66.3])</td><td>26/48 (54.2 [36.9 to 79.6])</td><td>29/42 (69.0 [48.0 to 99.4])</td><td>0.15</td><td>0.18<sup>#</sup></td></tr></tbody></table>

Values are quoted as the mean [95% CI] or no./total no. (rate [95%CI] in %). The *p* value was adjusted for the woman's age, BMI and current smoking status, the type of COH protocol, and the total dose of rFSH

<sup>f</sup> additionally adjusted for the number of fresh embryos transferred

<sup>#</sup> additionally adjusted for the total number of embryos transferred

Abbreviations: CPR clinical pregnancy rate, NA not applicable, due to low frequencies

<sup>‡</sup> cycles with no embryos obtained on day 2-3 were excluded

* cycles with embryo transfer on day 5-6 and cycles with a “freeze-all” strategy were excluded

## ICSI outcomes

After the exclusion of ICSI cycles requiring frozen-thawed sperm, the ICSI outcomes are shown in Table 6. There was no significant phenotype-related difference in the fertilization rate (percentages [95%CI] for A, C and D respectively: 61.8% (55.4 to 68.8%), 59.1% (52.0 to 67.2%), 59.2% (51.7 to 67.9%), adjusted *p*=0.93) or the mean number of day 2-3 embryos obtained (mean [95% CI]) for A, C and D respectively: 4.6 (4.1-5.1), 4.6 (4.1-5.3), 4.8 (4.2-5.5), *p*=0.84). The percentages of good quality (grade 1) embryos (percentage [95%CI] for A, C and D respectively: 49.8% (42.7 to 58.2%), 50.9% (42.3 to 61.2%), 51.7% (42.7 to 62.7%), adjusted *p*=0.86) and poor quality (grade 3) embryos (percentage [95%CI] for A, C and D respectively: 40.8% (34.3 to 48.4%), 40.1% (32.5 to 49.4%), 37.3% (29.7 to 46.8%), adjusted *p*=0.58) were also similar in the three phenotype groups. After adjustment for confounding factors we did not detect any intergroup differences in the IR, CPR, LBR, MR, and cumulative CPR. With regard

to ICSI outcomes, only the number of D2-D3 embryos and the number of frozen embryos were significantly higher in the PCOS group than in the PCOM and control groups (Supplemental Table 4).

## Discussion

Although a few published studies have investigated reproductive IVF/ICSI outcomes among women with PCOS, oocyte morphologic quality for the various PCOS phenotypes has not previously been described. Indeed, previous research by our group found that oocyte quality was similar in a PCOM group and a control women; however, the study's PCOM group contained a mixture of PCOM-only women and PCOS women [36]. It is well established that PCOM is a distinct phenotype and is not the same as PCOS [31]. We therefore decided to investigate oocyte morphology among the different PCOS phenotypes. To the best of our knowledge, the present study is the first to have addressed this topic. Our study population was homogenous and met strict inclusion
criteria - including ICSI for male infertility only. All cases with potential female infertility other than PCOS were excluded.

Importantly, we did not detect any significant differ-
ences in oocyte morphologic quality or embryo quality
between the PCOS phenotypes A, C and D. This unex-
pected observation challenges a number of preconceived
ideas. Firstly, some researchers have reported that rela-
tive to healthy women in IVF and ICSI programs, phe-
notypes A and B (considered to the most severe, with an
increased risk of comorbidities such as hyperinsulinism
and metabolic disorder) are associated with a lower preg-
nancy rate [18] and a greater risk of adverse outcomes
in pregnancy [16]. It has been suggested that these poor
outcomes are related to low oocyte quality or compe-
tence. Secondly, the cardinal features of PCOS (i.e. OA,
HA, and PCOM) are suspected to have an adverse impact
(independently or combined) on oocyte quality [15].
Indeed, androgens are involved in folliculogenesis, and a
hyperandrogenic environment leads to abnormal follicu-
logenesis, prematurely activated follicles, mitochondrial
abnormalities, and failure of meiosis progression to MII.
Furthermore, HA is known to induce premature luteini-
zation of the granulosa cells, which prevents them from
progressing to physiological atresia. It has been shown
that OA is associated with an alteration in the insulin
growth factor (IGF) pathway, which is involved in embry-
onic development and blastocyst formation [12]. How-
ever, the true extent of the IGF pathway's involvement in
the pathogenesis of PCOS is still unknown. Hence, the
impact of oligo-anovulation on oocyte quality and repro-
ductive outcomes (with the exception of the pregnancy
rate per cycle) has yet to be determined [15]. Likewise,
the impact of HA on oocyte quality is subject to debate.
Some researchers have highlighted a negative effect of
androgens on oocyte maturity in animal models [39], and
others have demonstrated the androgens' fundamental
role in early folliculogenesis and the pre-ovulatory folli-
cular stages [40]. The elevated androgen levels in PCOS
might lead to excess AMH secretion, which in turn is
involved in so-called "follicular arrest" in the ovaries [34,
41]. In contrast, Gaddas and colleagues (2016) did not
find any negative impact of biochemical HA on conven-
tional IVF or ICSI outcomes in women with PCOS [17].
Furthermore, a study by Palomba and colleagues (2010)
did not show any significant effects of clinically defined
HA and PCOM (PCOS phenotype C) [16], which is in
line with our present results. Lastly, our comparison of
oocyte quality in PCOS, PCOM-only and controls gave
much the same results as the only two other studies to
have evaluated morphologic abnormalities of the oocytes
[10, 36].

Our present results confirmed the above-mentioned
observation for clinical and endocrine features in PCOS
phenotype groups. Firstly, we did not find any differences
in the woman's BMI between the three A, C and D phe-
notypes - as also reported in a recent case-control study
[42]. Secondly, the PCOS phenotype A population exhib-
ited the highest AMH levels, which is in line with pre-
vious reports [18, 19, 42, 43]. Furthermore, the severity
of OA appeared to be correlated with an elevated serum
AMH level [44, 45]. We discovered that serum delta-
4-androstenedione levels were higher for PCOS phe-
notype A than for phenotype D, and that LH and AMH
levels were higher for phenotype A than for phenotypes
C and D. These results are also in line with another recent
report [19].

It is acknowledged that oocyte morphology is an
easily assessed, non-invasive marker of oocyte qual-
ity. Some oocyte abnormalities (i.e. a large perivitel-
line space, the presence of cytoplasmic vacuoles, or an
abnormal shape) are known to be associated with poor
reproductive outcomes [33, 37, 40, 41, 46]. In contrast,
some researchers did not find any differences in these
morphological parameters [34, 47, 48]. Likewise, our
study did not evidence an adverse relationship between
individual oocyte morphologic abnormalities and the
ICSI outcomes. Consequently, the oocyte morphol-
ogy's predictive value is still subject to debate [49-52].
We therefore assessed oocyte quality with regard to
not only the extra- and intra-cytoplasmic abnormali-
ties described above but also two scoring systems. Each
oocyte abnormality is considered separately in the
AOQI, [36], whereas the presence of specific oocyte
features are computed in the MOMS. In the present
study, we did not detect any differences between the
PCOS phenotypes A, C and D with regard to the AOQI
or the MOMS.

In line with previous reports of similar embryo qual-
ity in PCOS and non-PCOS patients [8, 10, 53, 54], we
did not observed interphenotype differences in these
variables or, indeed, in reproductive outcomes. This
contrast with recent reports in which PCOS A and B
were associated with a lower clinical pregnancy rate
[18] and PCOS phenotypes with HA were associated
with a lower cumulative live birth rates, when compared
with normo-androgenic counterparts [55]. This discrep-
ancy might be due to our strict inclusion criteria and
thus our small study population. Due to our relatively
small study sample size, we cannot exclude that several
associations were overlooked due to a lack of adequate
statistical power. For this reason, our results regarding
reproductive outcomes, should be interpreted with cau-
tion and furthers studies are warranted to confirm our
findings.
criteria - including ICSI for male infertility only. All cases with potential female infertility other than PCOS were excluded.

Importantly, we did not detect any significant differ-
ences in oocyte morphologic quality or embryo quality
between the PCOS phenotypes A, C and D. This unex-
pected observation challenges a number of preconceived
ideas. Firstly, some researchers have reported that rela-
tive to healthy women in IVF and ICSI programs, phe-
notypes A and B (considered to the most severe, with an
increased risk of comorbidities such as hyperinsulinism
and metabolic disorder) are associated with a lower preg-
nancy rate [18] and a greater risk of adverse outcomes
in pregnancy [16]. It has been suggested that these poor
outcomes are related to low oocyte quality or comppe-
tence. Secondly, the cardinal features of PCOS (i.e. OA,
HA, and PCOM) are suspected to have an adverse impact
(independently or combined) on oocyte quality [15].
Indeed, androgens are involved in folliculogenesis, and a
hyperandrogenic environment leads to abnormal follicu-
logenesis, prematurely activated follicles, mitochondrial
abnormalities, and failure of meiosis progression to MII.
Furthermore, HA is known to induce premature luteini-
zation of the granulosa cells, which prevents them from
progressing to physiological atresia. It has been shown
that OA is associated with an alteration in the insulin
growth factor (IGF) pathway, which is involved in embry-
onic development and blastocyst formation [12]. How-
ever, the true extent of the IGF pathway's involvement in
the pathogenesis of PCOS is still unknown. Hence, the
impact of oligo-anovulation on oocyte quality and repro-
ductive outcomes (with the exception of the pregnancy
rate per cycle) has yet to be determined [15]. Likewise,
the impact of HA on oocyte quality is subject to debate.
Some researchers have highlighted a negative effect of
androgens on oocyte maturity in animal models [39], and
others have demonstrated the androgens' fundamental
role in early folliculogenesis and the pre-ovulatory folli-
cular stages [40]. The elevated androgen levels in PCOS
might lead to excess AMH secretion, which in turn is
involved in so-called "follicular arrest" in the ovaries [34,
41]. In contrast, Gaddas and colleagues (2016) did not
find any negative impact of biochemical HA on conven-
tional IVF or ICSI outcomes in women with PCOS [17].
Furthermore, a study by Palomba and colleagues (2010)
did not show any significant effects of clinically defined
HA and PCOM (PCOS phenotype C) [16], which is in
line with our present results. Lastly, our comparison of
oocyte quality in PCOS, PCOM-only and controls gave
much the same results as the only two other studies to
have evaluated morphologic abnormalities of the oocytes
[10, 36].

Our present results confirmed the above-mentioned
observation for clinical and endocrine features in PCOS
phenotype groups. Firstly, we did not find any differences
in the woman's BMI between the three A, C and D phe-
notypes - as also reported in a recent case-control study
[42]. Secondly, the PCOS phenotype A population exhib-
ited the highest AMH levels, which is in line with pre-
vious reports [18, 19, 42, 43]. Furthermore, the severity
of OA appeared to be correlated with an elevated serum
AMH level [44, 45]. We discovered that serum delta-
4-androstenedione levels were higher for PCOS phe-
notype A than for phenotype D, and that LH and AMH
levels were higher for phenotype A than for phenotypes
C and D. These results are also in line with another recent
report [19].

It is acknowledged that oocyte morphology is an
easily assessed, non-invasive marker of oocyte qual-
ity. Some oocyte abnormalities (i.e. a large perivitel-
line space, the presence of cytoplasmic vacuoles, or an
abnormal shape) are known to be associated with poor
reproductive outcomes [33, 37, 40, 41, 46]. In contrast,
some researchers did not find any differences in these
morphological parameters [34, 47, 48]. Likewise, our
study did not evidence an adverse relationship between
individual oocyte morphologic abnormalities and the
ICSI outcomes. Consequently, the oocyte morphol-
ogy's predictive value is still subject to debate [49-52].
We therefore assessed oocyte quality with regard to
not only the extra- and intra-cytoplasmic abnormali-
ties described above but also two scoring systems. Each
oocyte abnormality is considered separately in the
AOQI, [36], whereas the presence of specific oocyte
features are computed in the MOMS. In the present
study, we did not detect any differences between the
PCOS phenotypes A, C and D with regard to the AOQI
or the MOMS.

In line with previous reports of similar embryo qual-
ity in PCOS and non-PCOS patients [8, 10, 53, 54], we
did not observed interphenotype differences in these
variables or, indeed, in reproductive outcomes. This
contrast with recent reports in which PCOS A and B
were associated with a lower clinical pregnancy rate
[18] and PCOS phenotypes with HA were associated
with a lower cumulative live birth rates, when compared
with normo-androgenic counterparts [55]. This discrep-
ancy might be due to our strict inclusion criteria and
thus our small study population. Due to our relatively
small study sample size, we cannot exclude that several
associations were overlooked due to a lack of adequate
statistical power. For this reason, our results regarding
reproductive outcomes, should be interpreted with cau-
tion and furthers studies are warranted to confirm our
findings.
Our study had a number of limitations. Firstly, we
cannot draw any conclusions with regard to the PCOS
phenotype B because we used the serum AMH level as
a surrogate marker of PCOM. In fact, our patients with
PCOS patients were diagnosed according to the revised
Rotterdam criteria [3, 25, 33]. Secondly, we used a
serum AMH threshold concentration > 35 pmol/l for
the PCOM group in our study [28, 29] This threshold
was established with an assay kit that is no longer com-
mercially available [56]. For consistency, the inclusion
period for the PCOS, PCOM-only and control groups
was restricted to the time when we used the AMH
assay; this also explains why our study population
was small. Some researchers consider that the serum
AMH level is a more reproducible, more sensitive vari-
able than the FNPO [16, 28, 29, 44, 57, 58]. The FNPO
threshold of 12 in the 2003 Rotterdam criteria is out-
dated and leads to overestimation of the prevalence
of PCOM in the general population [32, 59]. Conse-
quently, we decided to use the FNPO threshold of 19
reported in a cluster analysis [28]. This value is also
close to that given in international evidence-based
guidelines for the assessment and management of
PCOS from 2018 [4]. An AMH assay and pelvic ultra-
sound should be used together to determine the pres-
ence of PCOM and thus correct a false negative for one
or the other. Hence, their combined use, causes the
phenotype B to almost completely disappear [29]. Nev-
ertheless, we would like to point out that the results in
terms of pregnancy rates of ICSI cycles are presented
for information purposes only; our aim was to focus
on oocyte morphology as a possible marker of oocyte
quality in women with PCOS. Therefore, it is difficult
to compare the pregnancy rates results with previ-
ous studies, since our classification into A, B, C and D
phenotypes is also based on the result of AMH levels
[29], unlike those other studies [18, 55]. Indeed, in our
experience, a high AMH level with a specific threshold
adapted to the assay technique and determined in clus-
ter analysis can be considered as a biological equiva-
lent of PCOM as we previously published [28, 44, 56,
60]. Moreover, in these studies, several confounding
factors that could affect the results were not taken
into account in the statistical analysis, such as smok-
ing status or additional causes of women infertility
(endometriosis).

Secondly, our study's retrospective, single-center
design was associated with inherent bias. Nonetheless,
the single-center design enabled us to generate novel
data in a homogeneous patient population. All the
women received standardized care, and the proportion
of missing data was very low - constituting key study
strengths. Furthermore, we used very strict inclusion

and non-inclusion criteria, notably with regard to any
differential diagnoses; this also explains why the study
population was relatively small. Furthermore, we used
Poisson and logistic regressions to assess the effect of
potential confounding factors. One limitation of this
study was represented by the technical improvement
of ultrasound over the past 10 years which can impact
the antral follicle assessment, in particular regarding
small follicles. Nevertheless, our FNPO values were all
obtained with transvaginal ultrasound using a 5–9 mHz
probe, based on a routinely standardized protocol in
order to minimize the impact of technology improve-
ment on follicle counting.

Additionally, the type of gonadotropin used in ovarian stimulation in patients undergoing IVF/ICSI was not taken into account in the interpretation of oocyte morphology in PCOS women. Indeed, most of the data are rather in favor of an equal efficiency of the different types of gonadotropins during a COH protocol. The recent ESHRE recommendations concerning ovarian stimulation in IVF states that “The use of recombinant FSH (rFSH) and human menopausal gonadotropin (hMG) for ovarian stimulation is equally recommended” [61, 62]. However, some studies have assessed the impact of gonadotropin used for COH protocol in the oocyte and/or the embryo quality [63, 64]. The study by Ziebe et al. [63] has assessed the effects of gonadotropin type on early embryonic morphology up to day 3 of development, which appeared to be somewhat better with hMG, but it did not include women with PCOS. Indeed, only one study has assessed the impact of gonadotropin type used for COH protocol in women with PCOS [64], showing a significantly lower estradiol peak (E2), fewer intermediate-sized follicles, lower number of oocytes retrieved and MII oocytes in hMG-group in comparison with recombinant FSH-group. However, there were no significant differences in the number of fertilized oocytes, fertilization rates, top embryo quality, nor in the pregnancy rates between the two groups. Additionally, the authors did not assess the oocyte morphology.

Finally, we did not investigate whether the metabolic
profile (in particular insulin resistance and hyperinsuline-
mia) in PCOS women could impact oocyte morphology
in our study. Indeed, some authors have demonstrated
in a mice model that insulin resistance impaired both
oocyte quality and embryo development [65]. By con-
sequent, the comparison of detailed metabolic profile
in PCOS women in addition to the PCOS phenotype
(according Rotterdam) could be of interest in the evalu-
ation of oocyte quality and ICSI outcomes in futures pro-
spective studies.
Our study had a number of limitations. Firstly, we
cannot draw any conclusions with regard to the PCOS
phenotype B because we used the serum AMH level as
a surrogate marker of PCOM. In fact, our patients with
PCOS patients were diagnosed according to the revised
Rotterdam criteria [3, 25, 33]. Secondly, we used a
serum AMH threshold concentration > 35 pmol/l for
the PCOM group in our study [28, 29] This threshold
was established with an assay kit that is no longer com-
mercially available [56]. For consistency, the inclusion
period for the PCOS, PCOM-only and control groups
was restricted to the time when we used the AMH
assay; this also explains why our study population
was small. Some researchers consider that the serum
AMH level is a more reproducible, more sensitive vari-
able than the FNPO [16, 28, 29, 44, 57, 58]. The FNPO
threshold of 12 in the 2003 Rotterdam criteria is out-
dated and leads to overestimation of the prevalence
of PCOM in the general population [32, 59]. Conse-
quently, we decided to use the FNPO threshold of 19
reported in a cluster analysis [28]. This value is also
close to that given in international evidence-based
guidelines for the assessment and management of
PCOS from 2018 [4]. An AMH assay and pelvic ultra-
sound should be used together to determine the pres-
ence of PCOM and thus correct a false negative for one
or the other. Hence, their combined use, causes the
phenotype B to almost completely disappear [29]. Nev-
ertheless, we would like to point out that the results in
terms of pregnancy rates of ICSI cycles are presented
for information purposes only; our aim was to focus
on oocyte morphology as a possible marker of oocyte
quality in women with PCOS. Therefore, it is difficult
to compare the pregnancy rates results with previ-
ous studies, since our classification into A, B, C and D
phenotypes is also based on the result of AMH levels
[29], unlike those other studies [18, 55]. Indeed, in our
experience, a high AMH level with a specific threshold
adapted to the assay technique and determined in clus-
ter analysis can be considered as a biological equiva-
lent of PCOM as we previously published [28, 44, 56,
60]. Moreover, in these studies, several confounding
factors that could affect the results were not taken
into account in the statistical analysis, such as smok-
ing status or additional causes of women infertility
(endometriosis).

Secondly, our study's retrospective, single-center
design was associated with inherent bias. Nonetheless,
the single-center design enabled us to generate novel
data in a homogeneous patient population. All the
women received standardized care, and the proportion
of missing data was very low - constituting key study
strengths. Furthermore, we used very strict inclusion

and non-inclusion criteria, notably with regard to any
differential diagnoses; this also explains why the study
population was relatively small. Furthermore, we used
Poisson and logistic regressions to assess the effect of
potential confounding factors. One limitation of this
study was represented by the technical improvement
of ultrasound over the past 10 years which can impact
the antral follicle assessment, in particular regarding
small follicles. Nevertheless, our FNPO values were all
obtained with transvaginal ultrasound using a 5–9 mHz
probe, based on a routinely standardized protocol in
order to minimize the impact of technology improve-
ment on follicle counting.

Additionally, the type of gonadotropin used in ovarian stimulation in patients undergoing IVF/ICSI was not taken into account in the interpretation of oocyte morphology in PCOS women. Indeed, most of the data are rather in favor of an equal efficiency of the different types of gonadotropins during a COH protocol. The recent ESHRE recommendations concerning ovarian stimulation in IVF states that “The use of recombinant FSH (rFSH) and human menopausal gonadotropin (hMG) for ovarian stimulation is equally recommended” [61, 62]. However, some studies have assessed the impact of gonadotropin used for COH protocol in the oocyte and/or the embryo quality [63, 64]. The study by Ziebe et al. [63] has assessed the effects of gonadotropin type on early embryonic morphology up to day 3 of development, which appeared to be somewhat better with hMG, but it did not include women with PCOS. Indeed, only one study has assessed the impact of gonadotropin type used for COH protocol in women with PCOS [64], showing a significantly lower estradiol peak (E2), fewer intermediate-sized follicles, lower number of oocytes retrieved and MII oocytes in hMG-group in comparison with recombinant FSH-group. However, there were no significant differences in the number of fertilized oocytes, fertilization rates, top embryo quality, nor in the pregnancy rates between the two groups. Additionally, the authors did not assess the oocyte morphology.

Finally, we did not investigate whether the metabolic
profile (in particular insulin resistance and hyperinsuline-
mia) in PCOS women could impact oocyte morphology
in our study. Indeed, some authors have demonstrated
in a mice model that insulin resistance impaired both
oocyte quality and embryo development [65]. By con-
sequent, the comparison of detailed metabolic profile
in PCOS women in addition to the PCOS phenotype
(according Rotterdam) could be of interest in the evalu-
ation of oocyte quality and ICSI outcomes in futures pro-
spective studies.
In summary, our results showed that PCOS A, C and D phenotypes did not differ significantly with regard to oocyte morphologic quality and thus suggest that no single PCOS phenotype is associated with poor quality. Our results also suggest that the phenotype A (considered to be the most severe phenotype) is not associated with especially poor reproductive outcomes. Further studies are required to confirm these findings and characterize the underlying mechanisms.

**Abbreviations**

17-OHP: 17-hydroxyprogesterone; AMH: Anti-Müllerian hormone; AOQI: Average oocyte quality index; BMI: Body mass index; COH: Controlled ovarian hyperstimulation; CPR: Clinical pregnancy rate; E2: Estradiol; FNPO: Follicle number per ovary; FR: Fertilization rate; GEE: Generalized estimating equation; GnRH: Gonadotropin-releasing hormone; HA: Hyperandrogenism; hCG: Human chorionic gonadotropin; ICSI: Intracytoplasmic sperm injection; IQR: Interquartile range; IR: Implantation rate; IVF: In vitro fertilization; LBR: Live birth rate; MOMS: Metaphase (M)I oocyte morphologic score; MR: Miscarriage rate; OA: Oligo-ovulation/anovulation; PCOM: Polycystic ovarian morphology; PCOS: Polycystic ovary syndrome; PVS: Perivitelline space; TT: Total testosterone.

**Supplementary Information**

The online version contains supplementary material available at https://doi.org/10.1186/s12958-021-00874-2.

**Additional file 1.**

**Acknowledgements**

We thank David Fraser for revising the manuscript and English language editing.

**Authors' contributions**

A.U. contributed to manuscript drafting and critical discussion. C.G contributed to data acquisition. C.D, L.K and D.D. contributed to critical discussion. H.B. contributed to analysis of data. A.L.B. and G.R. contributed to study design, execution, acquisition of data, interpretation of data, manuscript drafting, and critical discussion. M.S. contributed to critical discussion and English language editing. All authors read and approved the final manuscript.

**Funding**

No external funding was used for the study.

**Availability of data and materials**

The data sets used and/or analyzed during the current study are available from the database of the Lille University Medical IVF center's on reasonable request.

**Declarations**

**Ethics approval and consent to participate**

Informed consent was obtained from all the couples. In line with the French legislation on retrospective studies of routine clinical practice, the study protocol was approved by a hospital committee with competency for research not requiring approval by an institutional review board.

**Consent for publication**

Not applicable.

**Competing interests**

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

**Author details**

¹CHU Lille, Institut de Biologie de la Reproduction-Spermiologie-CECOS, Jeanne de Flandre Hospital, Lille, France. ²Inserm UMR-S 1172, Laboratory of Development and Plasticity of the Neuroendocrine Brain, Lille, France.

³Department of Medicine, University of Lille, Lille, France. ⁴CHU Lille, Department of Endocrine Gynecology and Reproductive Medicine, Jeanne de Flandre Hospital, Lille, France. ⁵Inserm EA 4308 Gametogenèse et Qualité du Gamète, Institut de Biologie de la Reproduction-Spermiologie-CECOS, Lille, France. ⁶Univ. Lille, CHU Lille, ULR 2694 - METRICS: Évaluation des Technologies de Santé et des Pratiques Médicales, Lille, France.

Received: 27 July 2021 Accepted: 7 December 2021

Published online: 05 January 2022

**References**

1. Norman RJ, Dewailly D, Legro RS, Hickey TE. Polycystic ovary syndrome. Lancet. 2007;370(9588):685-97.

2. Lizneva D, Suturina L, Walker W, Brakta S, Gavrilova-Jordan L, Azziz R. Criteria, prevalence, and phenotypes of polycystic ovary syndrome. Fertil Steril. 2016;106(1):6-15.

3. ESHRE/ASRM-Sponsored PCOS Consensus Workshop Group. Revised 2003 consensus on diagnostic criteria and long-term health risks related to polycystic ovary syndrome. Fertil Steril. 2004;81(1):19-25.

4. Teede HJ, Misso ML, Costello MF, et al. Recommendations from the international evidence-based guideline for the assessment and management of polycystic ovary syndrome. Hum Reprod. 2018;33(9):1602-18.

5. Azziz R, Carmina E, Chen Z, et al. Polycystic ovary syndrome. Nat Rev Dis Primer. 2016;2:16057.

6. ESHRE/ASRM-Sponsored PCOS Consensus Workshop Group. Consensus on infertility treatment related to polycystic ovary syndrome. Hum Reprod. 2008;23(3):462-77.

7. Balen AH, Morley LC, Misso M, et al. The management of anovulatory infertility in women with polycystic ovary syndrome: an analysis of the evidence to support the development of global WHO guidance. Hum Reprod Update. 2016;22(6):687-708.

8. Ludwig M, Finas DF, Al-Hasani S, Diedrich K, Ortmann O. Oocyte quality and treatment outcome in intracytoplasmic sperm injection cycles of polycystic ovarian syndrome patients. Hum Reprod. 1999;14(2):354-8.

9. Heijnen EMEW, Eijkemans MJC, Hughes EG, Laven JSE, Macklon NS, Fauser BCJM. A meta-analysis of outcomes of conventional IVF in women with polycystic ovary syndrome. Hum Reprod Update. 2006;12(1):13-21.

10. Sahu B, Ozturk O, Ranierri M, Serhal P. Comparison of oocyte quality and intracytoplasmic sperm injection outcome in women with isolated polycystic ovaries or polycystic ovarian syndrome. Arch Gynecol Obstet. 2008;277(3):239-44.

11. Kollmann M, Martins WP, Lima MLS, et al. Strategies for improving outcome of assisted reproduction in women with polycystic ovary syndrome: systematic review and meta-analysis. Ultrasound Obstet Gynecol. 2016;48(6):709-18.

12. Qiao J, Feng HL. Extra- and intra-ovarian factors in polycystic ovary syndrome: impact on oocyte maturation and embryo developmental competence. Hum Reprod Update. 2011;17(1):17-33.

13. Sengoku K, Tamate K, Takuma N, Yoshida T, Goishi K, Ishikawa M. The chromosomal normality of unfertilized oocytes from patients with polycystic ovarian syndrome. Hum Reprod. 1997;12(3):474-7.

14. Li HWR, Lee VCY, Lau EYL, Yeung WSB, Ho PC, Ng EHY. Cumulative live-birth rate in women with polycystic ovary syndrome or isolated polycystic ovaries undergoing in-vitro fertilisation treatment. J Assist Reprod Genet. 2014;31(2):205-11.

15. Palomba S, Daolio J, La Sala GB. Oocyte competence in women with polycystic ovary syndrome. Trends Endocrinol Metab. 2017;28(3):186-98.

16. Palomba S, Falbo A, Russo T, Tolino A, Orio F, Zullo F. Pregnancy in women with polycystic ovary syndrome: the effect of different phenotypes
In summary, our results showed that PCOS A, C and D phenotypes did not differ significantly with regard to oocyte morphologic quality and thus suggest that no single PCOS phenotype is associated with poor quality. Our results also suggest that the phenotype A (considered to be the most severe phenotype) is not associated with especially poor reproductive outcomes. Further studies are required to confirm these findings and characterize the underlying mechanisms.

### Abbreviations

17-OHP: 17-hydroxyprogesterone; AMH: Anti-Müllerian hormone; AOQI: Average oocyte quality index; BMI: Body mass index; COH: Controlled ovarian hyperstimulation; CPR: Clinical pregnancy rate; E2: Estradiol; FNPO: Follicle number per ovary; FR: Fertilization rate; GEE: Generalized estimating equation; GnRH: Gonadotropin-releasing hormone; HA: Hyperandrogenism; hCG: Human chorionic gonadotropin; ICSI: Intracytoplasmic sperm injection; IQR: Interquartile range; IR: Implantation rate; IVF: In vitro fertilization; LBR: Live birth rate; MOMS: Metaphase (M) oocyte morphologic score; MR: Miscarriage rate; OA: Oligo-ovulation/anovulation; PCOM: Polycystic ovarian morphology; PCOS: Polycystic ovary syndrome; PVS: Perivitelline space; TT: Total testosterone.

## Supplementary Information

The online version contains supplementary material available at https://doi.org/10.1186/s12958-021-00874-2.

**Additional file 1.**

### Acknowledgements

We thank David Fraser for revising the manuscript and English language editing.

### Authors' contributions

A.U. contributed to manuscript drafting and critical discussion. C.G contributed to data acquisition. C.D, L.K and D.D. contributed to critical discussion. H.B. contributed to analysis of data. A.L.B. and G.R. contributed to study design, execution, acquisition of data, interpretation of data, manuscript drafting, and critical discussion. M.S. contributed to critical discussion and English language editing. All authors read and approved the final manuscript.

### Funding

No external funding was used for the study.

### Availability of data and materials

The data sets used and/or analyzed during the current study are available from the database of the Lille University Medical IVF center's on reasonable request.

## Declarations

### Ethics approval and consent to participate

Informed consent was obtained from all the couples. In line with the French legislation on retrospective studies of routine clinical practice, the study protocol was approved by a hospital committee with competency for research not requiring approval by an institutional review board.

### Consent for publication

Not applicable.

## Competing interests

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

## Author details

¹CHU Lille, Institut de Biologie de la Reproduction-Spermiologie-CECOS, Jeanne de Flandre Hospital, Lille, France. ²Inserm UMR-S 1172, Laboratory of Development and Plasticity of the Neuroendocrine Brain, Lille, France.

³Department of Medicine, University of Lille, Lille, France. ⁴CHU Lille, Department of Endocrine Gynecology and Reproductive Medicine, Jeanne de Flandre Hospital, Lille, France. ⁵Inserm EA 4308 Gametogenèse et Qualité du Gamète, Institut de Biologie de la Reproduction-Spermiologie-CECOS, Lille, France. ⁶Univ. Lille, CHU Lille, ULR 2694 - METRICS: Évaluation des Technologies de Santé et des Pratiques Médicales, Lille, France.

Received: 27 July 2021 Accepted: 7 December 2021

Published online: 05 January 2022

## References

1. Norman RJ, Dewailly D, Legro RS, Hickey TE. Polycystic ovary syndrome. Lancet. 2007;370(9588):685-97.

2. Lizneva D, Suturina L, Walker W, Brakta S, Gavrilova-Jordan L, Azziz R. Criteria, prevalence, and phenotypes of polycystic ovary syndrome. Fertil Steril. 2016;106(1):6-15.

3. ESHRE/ASRM-Sponsored PCOS Consensus Workshop Group. Revised 2003 consensus on diagnostic criteria and long-term health risks related to polycystic ovary syndrome. Fertil Steril. 2004;81(1):19-25.

4. Teede HJ, Misso ML, Costello MF, et al. Recommendations from the international evidence-based guideline for the assessment and management of polycystic ovary syndrome. Hum Reprod. 2018;33(9):1602-18.

5. Azziz R, Carmina E, Chen Z, et al. Polycystic ovary syndrome. Nat Rev Dis Primer. 2016;2:16057.

6. ESHRE/ASRM-Sponsored PCOS Consensus Workshop Group. Consensus on infertility treatment related to polycystic ovary syndrome. Hum Reprod. 2008;23(3):462-77.

7. Balen AH, Morley LC, Misso M, et al. The management of anovulatory infertility in women with polycystic ovary syndrome: an analysis of the evidence to support the development of global WHO guidance. Hum Reprod Update. 2016;22(6):687-708.

8. Ludwig M, Finas DF, Al-Hasani S, Diedrich K, Ortmann O. Oocyte quality and treatment outcome in intracytoplasmic sperm injection cycles of polycystic ovarian syndrome patients. Hum Reprod. 1999;14(2):354-8.

9. Heijnen EMEW, Eijkemans MJC, Hughes EG, Laven JSE, Macklon NS, Fauser BCJM. A meta-analysis of outcomes of conventional IVF in women with polycystic ovary syndrome. Hum Reprod Update. 2006;12(1):13-21.

10. Sahu B, Ozturk O, Ranierri M, Serhal P. Comparison of oocyte quality and intracytoplasmic sperm injection outcome in women with isolated polycystic ovaries or polycystic ovarian syndrome. Arch Gynecol Obstet. 2008;277(3):239-44.

11. Kollmann M, Martins WP, Lima MLS, et al. Strategies for improving outcome of assisted reproduction in women with polycystic ovary syndrome: systematic review and meta-analysis. Ultrasound Obstet Gynecol. 2016;48(6):709-18.

12. Qiao J, Feng HL. Extra- and intra-ovarian factors in polycystic ovary syndrome: impact on oocyte maturation and embryo developmental competence. Hum Reprod Update. 2011;17(1):17-33.

13. Sengoku K, Tamate K, Takuma N, Yoshida T, Goishi K, Ishikawa M. The chromosomal normality of unfertilized oocytes from patients with polycystic ovarian syndrome. Hum Reprod. 1997;12(3):474-7.

14. Li HWR, Lee VCY, Lau EYL, Yeung WSB, Ho PC, Ng EHY. Cumulative live-birth rate in women with polycystic ovary syndrome or isolated polycystic ovaries undergoing in-vitro fertilisation treatment. J Assist Reprod Genet. 2014;31(2):205-11.

15. Palomba S, Daolio J, La Sala GB. Oocyte competence in women with polycystic ovary syndrome. Trends Endocrinol Metab. 2017;28(3):186-98.

16. Palomba S, Falbo A, Russo T, Tolino A, Orio F, Zullo F. Pregnancy in women with polycystic ovary syndrome: the effect of different phenotypes
and features on obstetric and neonatal outcomes. Fertil Steril.
2010;94(5):1805-11.

17. Gaddas M, Chaouache N, Ajina M, Tabka Z, Zaouali AM. Does Hyperandrogenic statute in the polycystic ovary syndrome constitute an obstacle to the success of in vitro fertilization? J Gynecol Obstet Biol Reprod. 2016;45(9):1091-8.

18. Ramezanali F, Ashrafی M, Hemat M, Arabipoor A, Jalali S, Moini A. Assisted reproductive outcomes in women with different polycystic ovary syndrome phenotypes: the predictive value of anti-Müllerian hormone. Reprod BioMed Online. 2016;32(5):503-12.

19. Cela V, Obino MER, Alberga Y, et al. Ovarian response to controlled ovarian stimulation in women with different polycystic ovary syndrome phenotypes. Gynecol Endocrinol. 2018;34(6):518-23.

20. Dumesic DA, Oberfield SE, Stener-Victorin E, Marshall JC, Laven JS, Legro RS. Scientific statement on the diagnostic criteria, epidemiology, pathophysiology, and molecular genetics of polycystic ovary syndrome. Endocr Rev. 2015;36(5):487-525.

21. Alikani M, Palermo G, Adler A, Bertoli M, Blake M, Cohen J. Intracytoplasmic sperm injection in dysmorphic human oocytes. Zygote. 1995;3(4):283-8.

22. Kahraman S, Yakin K, Dönmez E, et al. Relationship between granular cytoplasm of oocytes and pregnancy outcome following intracytoplasmic sperm injection. Hum Reprod. 2000;15(11):2390-3.

23. Gilchrist RB, Lane M, Thompson JG. Oocyte-secreted factors: regulators of cumulus cell function and oocyte quality. Hum Reprod Update. 2008;14(2):159-77.

24. Ebner T, Moser M, Tews G. Is oocyte morphology prognostic of embryo developmental potential after ICSI? Reprod BioMed Online. 2006;12(4):507-12.

25. Merviel P, Cabry R, Chardon K, et al. Impact of oocytes with CLCG on ICSI outcomes and their potential relation to pesticide exposure. J Ovarian Res. 2017;10(1):42.

26. von Elm E, Altman DG, Egger M, et al. The strengthening the reporting of observational studies in epidemiology (STROBE) statement: guidelines for reporting observational studies. Lancet. 2007;370(9596):1453-7.

27. Ferraretti AP, La Marca A, Fauser BCJM, Tarlatzis B, Nargund G, Gianaroli L. ESHRE consensus on the definition of 'poor response' to ovarian stimulation for in vitro fertilization: the Bologna criteria. Hum Reprod. 2011;26(7):1616-24.

28. Dewailly D, Gronier H, Poncelet E, et al. Diagnosis of polycystic ovary syndrome (PCOS): revisiting the threshold values of follicle count on ultrasound and of the serum AMH level for the definition of polycystic ovaries. Hum Reprod. 2011;26(11):3123-9.

29. Fraissinet A, Robin G, Pigny P, Lefebvre T, Catteau-Jonard S, Dewailly D. Use of the serum anti-Müllerian hormone assay as a surrogate for polycystic ovarian morphology: impact on diagnosis and phenotypic classification of polycystic ovary syndrome. Hum Reprod. 2017;32(8):1716-22.

30. Escobar-Morreale HF, Carmina E, Dewailly D, et al. Epidemiology, diagnosis and management of hirsutism: a consensus statement by the androgen excess and polycystic ovary syndrome society. Hum Reprod Update. 2012;18(2):146-70.

31. Catteau-Jonard S, Bancquart J, Poncelet E, Lefebvre-Maunoury C, Robin G, Dewailly D. Polycystic ovaries at ultrasound: normal variant or silent polycystic ovary syndrome? Ultrasound Obstet Gynecol. 2012;40(2):223-9.

32. Dewailly D. Diagnostic criteria for PCOS: is there a need for a rethink? Best Pract Res Clin Obstet Gynaecol. 2016;37:5-11.

33. Pigny P, Merlen E, Robert Y, et al. Elevated serum level of anti-mullerian hormone in patients with polycystic ovary syndrome: relationship to the ovarian follicle excess and to the follicular arrest. J Clin Endocrinol Metab. 2003;88(12):5957-62.

34. Jonard S, Dewailly D. The follicular excess in polycystic ovaries, due to intra-ovarian hyperandrogenism, may be the main culprit for the follicular arrest. Hum Reprod Update. 2004;10(2):107-17.

35. Alpha Scientists in Reproductive Medicine and ESHRE Special Interest Group of Embryology. The Istanbul consensus workshop on embryo assessment: proceedings of an expert meeting. Hum Reprod. 2011;26(6):1270-83.

36. Sigala J, Sifer C, Dewailly D, et al. Is polycystic ovarian morphology related to a poor oocyte quality after controlled ovarian hyperstimulation for intracytoplasmic sperm injection? Results from a prospective, comparative study. Fertil Steril. 2015;103(1):112-8. https://doi.org/10.1016/j.fertnstert.2014.09.040.

37. Rienzi L, Ubaldi FM, Iacobelli M, et al. Significance of metaphase II human oocyte morphology on ICSI outcome. Fertil Steril. 2008;90(5):1692-700.

38. Van Steirteghem AC, Nagy Z, Joris H, et al. High fertilization and implantation rates after intracytoplasmic sperm injection. Hum Reprod. 1993;8(7):1061-6.

39. Anderiez C, Trounson AO. The effect of testosterone on the maturation and developmental capacity of murine oocytes in vitro. Hum Reprod. 1995;10(9):2377-81.

40. Hu Y-C, Wang P-H, Yeh S, et al. Subfertility and defective folliculogenesis in female mice lacking androgen receptor. Proc Natl Acad Sci U S A. 2004;101(31):11209-14.

41. Dewailly D, Robin G, Peigne M, Decanter C, Pigny P, Catteau-Jonard S. Interactions between androgens, FSH, anti-Müllerian hormone and estradiol during folliculogenesis in the human normal and polycystic ovary. Hum Reprod Update. 2016;22(6):709-24.

42. Jamil AS, Alalaf SK, Al-Tawil NG, Al-Shawaf T. Comparison of clinical and hormonal characteristics among four phenotypes of polycystic ovary syndrome based on the Rotterdam criteria. Arch Gynecol Obstet. 2016;293(2):447-56.

43. Piouka A, Farmakiotis D, Katsikis I, Macut D, Gerou S, Panidis D. Anti-Mullerian hormone levels reflect severity of PCOS but are negatively influenced by obesity: relationship with increased luteinizing hormone levels. Am J Physiol Endocrinol Metab. 2009;296(2):E238-43.

44. Pigny P, Jonard S, Robert Y, Dewailly D. Serum anti-Müllerian hormone as a surrogate for Antral follicle count for definition of the polycystic ovary syndrome. J Clin Endocrinol Metab. 2006;91(3):941-5.

45. Bhide P, Kulkarni A, Dilgil M, et al. Phenotypic variation in anti-Mullerian hormone (AMH) production per follicle in women with polycystic ovary syndrome (PCOS) and isolated polycystic ovarian morphology (PCOM): an observational cross-sectional study. Gynecol Endocrinol. 2017;33(10):801-6.

46. Braga DPAF, Setti AS, de Figueira RCS, Machado RB, Iaconelli A, Borges E. Influence of oocyte dysmorphisms on blastocyst formation and quality. Fertil Steril. 2013;100(3):748-54.

47. Sauerbrun-Cutler M-T, Vega M, Breborowicz A, et al. Oocyte zona pelúcida dysmorphology is associated with diminished in-vitro fertilization success. J Ovarian Res. 2015;8:5.

48. Dal Canto M, Guglielmo MC, Mignini Renzini M, et al. Dysmorphic patterns are associated with cytoskeletal alterations in human oocytes. Hum Reprod. 2017;32(4):750-7.

49. De Sutter P, Dozortsev D, Qian C, Dhont M. Oocyte morphology does not correlate with fertilization rate and embryo quality after intracytoplasmic sperm injection. Hum Reprod. 1996;11(3):595-7.

50. Balaban B, Urman B, Sertac A, Alatas C, Aksoy S, Mercan R. Oocyte morphology does not affect fertilization rate, embryo quality and implantation rate after intracytoplasmic sperm injection. Hum Reprod. 1998;13(12):3431-3.

51. Setti AS, Figueira RCS, Braga DPAF, Colturato SS, Iaconelli A, Borges E. Relationship between oocyte abnormal morphology and intracytoplasmic sperm injection outcomes: a meta-analysis. Eur J Obstet Gynecol Reprod Biol. 2011;159(2):364-70.

52. Yu EJ, Ahn H, Lee JM, Jee BC, Kim SH. Fertilization and embryo quality of mature oocytes with specific morphologic abnormalities. Clin Exp Reprod Med. 2015;42(4):156-62.

53. Esinler I, Bayar U, Bozdağ G, Yarali H. Outcome of intracytoplasmic sperm injection in patients with polycystic ovary syndrome or isolated polycystic ovaries. Fertil Steril. 2005;84(4):932-7.

54. Kdous M, Chaker A, Zhioua A, Zhioua F. Oocyte and Embryo Quality and Outcome of ICSI Cycles in Patients With Polycystic Ovary Syndrome (PCOS) Versus Normo-Ovulatory. J Gynécologie Obstétrique Biol Reprod. 2009;38(2):133-43 Published online March 30, 2009.

55. De Vos M, Pareyn S, Drakopoulos P, et al. Cumulative live birth rates after IVF in patients with polycystic ovaries: phenotype matters. Reprod BioMed Online. 2018;37(2):163-71.

56. Pigny P, Gorisse E, Ghulam A, Robin G, Catteau-Jonard S, Duhamel A, et al. Comparative assessment of five serum antimüllerian hormone assays for the diagnosis of polycystic ovary syndrome. Fertil Steril. 2016;105(4):1063-1069.e3.
and features on obstetric and neonatal outcomes. Fertil Steril.
2010;94(5):1805-11.

17. Gaddas M, Chaouache N, Ajina M, Tabka Z, Zaouali AM. Does Hyperandrogenic statute in the polycystic ovary syndrome constitute an obstacle to the success of in vitro fertilization? J Gynecol Obstet Biol Reprod. 2016;45(9):1091-8.

18. Ramezanali F, Ashrafی M, Hemat M, Arabipoor A, Jalali S, Moini A. Assisted reproductive outcomes in women with different polycystic ovary syndrome phenotypes: the predictive value of anti-Müllerian hormone. Reprod BioMed Online. 2016;32(5):503-12.

19. Cela V, Obino MER, Alberga Y, et al. Ovarian response to controlled ovarian stimulation in women with different polycystic ovary syndrome phenotypes. Gynecol Endocrinol. 2018;34(6):518-23.

20. Dumesic DA, Oberfield SE, Stener-Victorin E, Marshall JC, Laven JS, Legro RS. Scientific statement on the diagnostic criteria, epidemiology, pathophysiology, and molecular genetics of polycystic ovary syndrome. Endocr Rev. 2015;36(5):487-525.

21. Alikani M, Palermo G, Adler A, Bertoli M, Blake M, Cohen J. Intracytoplasmic sperm injection in dysmorphic human oocytes. Zygote. 1995;3(4):283-8.

22. Kahraman S, Yakin K, Dönmez E, et al. Relationship between granular cytoplasm of oocytes and pregnancy outcome following intracytoplasmic sperm injection. Hum Reprod. 2000;15(11):2390-3.

23. Gilchrist RB, Lane M, Thompson JG. Oocyte-secreted factors: regulators of cumulus cell function and oocyte quality. Hum Reprod Update. 2008;14(2):159-77.

24. Ebner T, Moser M, Tews G. Is oocyte morphology prognostic of embryo developmental potential after ICSI? Reprod BioMed Online. 2006;12(4):507-12.

25. Merviel P, Cabry R, Chardon K, et al. Impact of oocytes with CLCG on ICSI outcomes and their potential relation to pesticide exposure. J Ovarian Res. 2017;10(1):42.

26. von Elm E, Altman DG, Egger M, et al. The strengthening the reporting of observational studies in epidemiology (STROBE) statement: guidelines for reporting observational studies. Lancet. 2007;370(9596):1453-7.

27. Ferraretti AP, La Marca A, Fauser BCJM, Tarlatzis B, Nargund G, Gianaroli L. ESHRE consensus on the definition of 'poor response' to ovarian stimulation for in vitro fertilization: the Bologna criteria. Hum Reprod. 2011;26(7):1616-24.

28. Dewailly D, Gronier H, Poncelet E, et al. Diagnosis of polycystic ovary syndrome (PCOS): revisiting the threshold values of follicle count on ultrasound and of the serum AMH level for the definition of polycystic ovaries. Hum Reprod. 2011;26(11):3123-9.

29. Fraissinet A, Robin G, Pigny P, Lefebvre T, Catteau-Jonard S, Dewailly D. Use of the serum anti-Müllerian hormone assay as a surrogate for polycystic ovarian morphology: impact on diagnosis and phenotypic classification of polycystic ovary syndrome. Hum Reprod. 2017;32(8):1716-22.

30. Escobar-Morreale HF, Carmina E, Dewailly D, et al. Epidemiology, diagnosis and management of hirsutism: a consensus statement by the androgen excess and polycystic ovary syndrome society. Hum Reprod Update. 2012;18(2):146-70.

31. Catteau-Jonard S, Bancquart J, Poncelet E, Lefebvre-Maunoury C, Robin G, Dewailly D. Polycystic ovaries at ultrasound: normal variant or silent polycystic ovary syndrome? Ultrasound Obstet Gynecol. 2012;40(2):223-9.

32. Dewailly D. Diagnostic criteria for PCOS: is there a need for a rethink? Best Pract Res Clin Obstet Gynaecol. 2016;37:5-11.

33. Pigny P, Merlen E, Robert Y, et al. Elevated serum level of anti-mullerian hormone in patients with polycystic ovary syndrome: relationship to the ovarian follicle excess and to the follicular arrest. J Clin Endocrinol Metab. 2003;88(12):5957-62.

34. Jonard S, Dewailly D. The follicular excess in polycystic ovaries, due to intra-ovarian hyperandrogenism, may be the main culprit for the follicular arrest. Hum Reprod Update. 2004;10(2):107-17.

35. Alpha Scientists in Reproductive Medicine and ESHRE Special Interest Group of Embryology. The Istanbul consensus workshop on embryo assessment: proceedings of an expert meeting. Hum Reprod. 2011;26(6):1270-83.

36. Sigala J, Sifer C, Dewailly D, et al. Is polycystic ovarian morphology related to a poor oocyte quality after controlled ovarian hyperstimulation for intracytoplasmic sperm injection? Results from a prospective, comparative study. Fertil Steril. 2015;103(1):112-8. https://doi.org/10.1016/j.fertnstert.2014.09.040.

37. Rienzi L, Ubaldi FM, Iacobelli M, et al. Significance of metaphase II human oocyte morphology on ICSI outcome. Fertil Steril. 2008;90(5):1692-700.

38. Van Steirteghem AC, Nagy Z, Joris H, et al. High fertilization and implantation rates after intracytoplasmic sperm injection. Hum Reprod. 1993;8(7):1061-6.

39. Anderiesz C, Trounson AO. The effect of testosterone on the maturation and developmental capacity of murine oocytes in vitro. Hum Reprod. 1995;10(9):2377-81.

40. Hu Y-C, Wang P-H, Yeh S, et al. Subfertility and defective folliculogenesis in female mice lacking androgen receptor. Proc Natl Acad Sci U S A. 2004;101(31):11209-14.

41. Dewailly D, Robin G, Peigne M, Decanter C, Pigny P, Catteau-Jonard S. Interactions between androgens, FSH, anti-Müllerian hormone and estradiol during folliculogenesis in the human normal and polycystic ovary. Hum Reprod Update. 2016;22(6):709-24.

42. Jamil AS, Alalaf SK, Al-Tawil NG, Al-Shawaf T. Comparison of clinical and hormonal characteristics among four phenotypes of polycystic ovary syndrome based on the Rotterdam criteria. Arch Gynecol Obstet. 2016;293(2):447-56.

43. Piouka A, Farmakiotis D, Katsikis I, Macut D, Gerou S, Panidis D. Anti-Mullerian hormone levels reflect severity of PCOS but are negatively influenced by obesity: relationship with increased luteinizing hormone levels. Am J Physiol Endocrinol Metab. 2009;296(2):E238-43.

44. Pigny P, Jonard S, Robert Y, Dewailly D. Serum anti-Müllerian hormone as a surrogate for Antral follicle count for definition of the polycystic ovary syndrome. J Clin Endocrinol Metab. 2006;91(3):941-5.

45. Bhide P, Kulkarni A, Dilgil M, et al. Phenotypic variation in anti-Mullerian hormone (AMH) production per follicle in women with polycystic ovary syndrome (PCOS) and isolated polycystic ovarian morphology (PCOM): an observational cross-sectional study. Gynecol Endocrinol. 2017;33(10):801-6.

46. Braga DPAF, Setti AS, de Figueira RCS, Machado RB, Iaconelli A, Borges E. Influence of oocyte dysmorphisms on blastocyst formation and quality. Fertil Steril. 2013;100(3):748-54.

47. Sauerbrun-Cutler M-T, Vega M, Breborowicz A, et al. Oocyte zona pelúcida dysmorphology is associated with diminished in-vitro fertilization success. J Ovarian Res. 2015;8:5.

48. Dal Canto M, Guglielmo MC, Mignini Renzini M, et al. Dysmorphic patterns are associated with cytoskeletal alterations in human oocytes. Hum Reprod. 2017;32(4):750-7.

49. De Sutter P, Dozortsev D, Qian C, Dhont M. Oocyte morphology does not correlate with fertilization rate and embryo quality after intracytoplasmic sperm injection. Hum Reprod. 1996;11(3):595-7.

50. Balaban B, Urman B, Sertac A, Alatas C, Aksoy S, Mercan R. Oocyte morphology does not affect fertilization rate, embryo quality and implantation rate after intracytoplasmic sperm injection. Hum Reprod. 1998;13(12):3431-3.

51. Setti AS, Figueira RCS, Braga DPAF, Colturato SS, Iaconelli A, Borges E. Relationship between oocyte abnormal morphology and intracytoplasmic sperm injection outcomes: a meta-analysis. Eur J Obstet Gynecol Reprod Biol. 2011;159(2):364-70.

52. Yu EJ, Ahn H, Lee JM, Jee BC, Kim SH. Fertilization and embryo quality of mature oocytes with specific morphologic abnormalities. Clin Exp Reprod Med. 2015;42(4):156-62.

53. Esinler I, Bayar U, Bozdag G, Yarali H. Outcome of intracytoplasmic sperm injection in patients with polycystic ovary syndrome or isolated polycystic ovaries. Fertil Steril. 2005;84(4):932-7.

54. Kdous M, Chaker A, Zhioua A, Zhioua F. Oocyte and Embryo Quality and Outcome of ICSI Cycles in Patients With Polycystic Ovary Syndrome (PCOS) Versus Normo-Ovulatory. J Gynécologie Obstétrique Biol Reprod. 2009;38(2):133-43 Published online March 30, 2009.

55. De Vos M, Pareyn S, Drakopoulos P, et al. Cumulative live birth rates after IVF in patients with polycystic ovaries: phenotype matters. Reprod BioMed Online. 2018;37(2):163-71.

56. Pigny P, Gorisse E, Ghulam A, Robin G, Catteau-Jonard S, Duhamel A, et al. Comparative assessment of five serum antimüllerian hormone assays for the diagnosis of polycystic ovary syndrome. Fertil Steril. 2016;105(4):1063-1069.e3.
57. Iliodromiti S, Kelsey TW, Anderson RA, Nelson SM. Can anti-Mullerian hormone predict the diagnosis of polycystic ovary syndrome? A systematic review and meta-analysis of extracted data. J Clin Endocrinol Metab. 2013;98(8):3332–40.

58. Lauritsen MP, Bentzen JG, Pinborg A, et al. The prevalence of polycystic ovary syndrome in a normal population according to the Rotterdam criteria versus revised criteria including anti-Mullerian hormone. Hum Reprod. 2014;29(4):791–801.

59. Dewailly D, Lujan ME, Carmina E, et al. Definition and significance of polycystic ovarian morphology: a task force report from the androgen excess and polycystic ovary syndrome society. Hum Reprod Update. 2014;20(3):334–52.

60. Robin G, Gallo C, Catteau-Jonard S, Lefebvre-Maunoury C, Pigny P, Duhamel A, et al. Polycystic ovary-like abnormalities (PCO-L) in women with functional hypothalamic amenorrhea. J Clin Endocrinol Metab. 2012;97(11):4236–43.

61. Ovarian Stimulation TEGGO, et al. ESHRE guideline: ovarian stimulation for IVF/ICSI. Hum Reprod Open. 2020;2020(2):hoaa009.

62. van Wely M, Kwan I, Burt AL, Thomas J, Vail A, Van der Veen F, et al. Recombinant versus urinary gonadotrophin for ovarian stimulation in assisted reproductive technology cycles. Cochrane Database Syst Rev. 2011;2011(2):Cd005354.

63. Ziebe S, Lundin K, Janssens R, Helmgaard L, Arce JC. MERIT (Menotrophin vs recombinant FSH in vitro fertilisation trial) group. Influence of ovarian stimulation with HP-hMG or recombinant FSH on embryo quality parameters in patients undergoing IVF. Hum Reprod. 2007;22(9):2404–13.

64. Figen Turkcapar A, Seckin B, Onalan G, Ozdener T, Batioglu S. Human menopausal gonadotropin versus recombinant FSH in polycystic ovary syndrome patients undergoing in vitro fertilization. Int J Fertil Steril. 2013;6(4):238–43.

65. Ou XH, Li S, Wang ZB, Li M, Quan S, Xing F, et al. Maternal insulin resistance causes oxidative stress and mitochondrial dysfunction in mouse oocytes. Hum Reprod. 2012;27(7):2130–45.

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<figure><img src="image_3.png" /></figure>
57. Iliodromiti S, Kelsey TW, Anderson RA, Nelson SM. Can anti-Mullerian hormone predict the diagnosis of polycystic ovary syndrome? A systematic review and meta-analysis of extracted data. J Clin Endocrinol Metab. 2013;98(8):3332–40.

58. Lauritsen MP, Bentzen JG, Pinborg A, et al. The prevalence of polycystic ovary syndrome in a normal population according to the Rotterdam criteria versus revised criteria including anti-Mullerian hormone. Hum Reprod. 2014;29(4):791–801.

59. Dewailly D, Lujan ME, Carmina E, et al. Definition and significance of polycystic ovarian morphology: a task force report from the androgen excess and polycystic ovary syndrome society. Hum Reprod Update. 2014;20(3):334–52.

60. Robin G, Gallo C, Catteau-Jonard S, Lefebvre-Maunoury C, Pigny P, Duhamel A, et al. Polycystic ovary-like abnormalities (PCO-L) in women with functional hypothalamic amenorrhea. J Clin Endocrinol Metab. 2012;97(11):4236–43.

61. Ovarian Stimulation TEGGO, et al. ESHRE guideline: ovarian stimulation for IVF/ICSI. Hum Reprod Open. 2020;2020(2):hoaa009.

62. van Wely M, Kwan I, Burt AL, Thomas J, Vail A, Van der Veen F, et al. Recombinant versus urinary gonadotrophin for ovarian stimulation in assisted reproductive technology cycles. Cochrane Database Syst Rev. 2011;2011(2):Cd005354.

63. Ziebe S, Lundin K, Janssens R, Helmgaard L, Arce JC. MERIT (Menotrophin vs recombinant FSH in vitro fertilisation trial) group. Influence of ovarian stimulation with HP-hMG or recombinant FSH on embryo quality parameters in patients undergoing IVF. Hum Reprod. 2007;22(9):2404–13.

64. Figen Turkcapar A, Seckin B, Onalan G, Ozdener T, Batioglu S. Human menopausal gonadotropin versus recombinant FSH in polycystic ovary syndrome patients undergoing in vitro fertilization. Int J Fertil Steril. 2013;6(4):238–43.

65. Ou XH, Li S, Wang ZB, Li M, Quan S, Xing F, et al. Maternal insulin resistance causes oxidative stress and mitochondrial dysfunction in mouse oocytes. Hum Reprod. 2012;27(7):2130–45.

**Publisher's Note**

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Ready to submit your research? Choose BMC and benefit from:

* fast, convenient online submission

* thorough peer review by experienced researchers in your field

* rapid publication on acceptance

* support for research data, including large and complex data types

* gold Open Access which fosters wider collaboration and increased citations

* maximum visibility for your research: over 100M website views per year

At BMC, research is always in progress.

Learn more biomedcentral.com/submissions

<figure><img src="image_4.png" /></figure>